Folate Conjugates

ABSTRACT

The present invention provides iRNA agent including at least one monomer having the structure shown in formula (I′) 
     
       
         
         
             
             
         
       
     
     wherein:
         A and B are each independently for each occurrence O, N(R N ) or S;   X is H, a protecting group, a phosphate group, a phosphodiester group, an activated phosphate group, an activated phosphite group, a phosphoramidite, a solid support, —P(Z′)(Z″)O-nucleoside, —P(Z′)(Z″)O-oligonucleotide, a lipid, a PEG, a steroid, a polymer, —P(Z′)(Z″)O-L 6 -Q′-L 7 -OP(Z′″)(Z″″)O-oligonucleotide, a nucleotide, or an oligonucleotide;   Y is H, a protecting group, a phosphate group, a phosphodiester group, an activated phosphate group, an activated phosphite group, a phosphoramidite, a solid support, —P(Z′)(Z″)O-nucleoside, —P(Z′)(Z″)O-oligonucleotide, a lipid, a PEG, a steroid, a lipophile, a polymer, —P(Z′)(Z″)O-L 6 -Q′-L 7 -OP(Z′″)(Z″″)O-oligonucleotide, a nucleotide, or an oligonucleotide;   R is folate, a folate analog a folate mimic or a folate receptor binding ligand;   L 6  and L 7  are each independently for each occurrence —(CH 2 ) n —, —C(R′)(R″)(CH 2 ) n —, —(CH 2 ) n C(R′)(R″)—, —(CH 2 CH 2 O) m CH 2 CH 2 —, or —(CH 2 CH 2 O) m CH 2 CH 2 NH—;   Q′ is NH, O, S, CH 2 , C(O)O, C(O)NH, —NH—CH(R a )—C(O)—, —C(O)—CH(R a )—NH—, CO,       

     
       
         
         
             
             
         
       
         
         
           
              where R a  is H or amino acid side; chain. 
             R′ and R″ are each independently H, CH 3 , OH, SH, NH 2 , NH(Alkyl=Me, Et, Pr, isoPr, Bu, Bn) or N(diAlkyl=Me 2 , Et 2 , Bn 2 ); 
             Z′, Z″, Z′″ and Z″″ are independently O or S; 
             n represent independently for each occurrence 1-20; and 
             m represent independently for each occurrence 0-50.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/328,537, filed Dec. 4, 2008, which claims the benefit of priority toU.S. Provisional Patent Application Ser. No. 60/992,309, filed Dec. 4,2007; and U.S. Provisional Patent Application Ser. No. 61/013,597 filedDec. 13, 2007, all of which are incorporated herein by reference intheir entirety.

GOVERNMENT SUPPORT

The work described herein was carried out, at least in part, using fundsfrom the United States government under contract numberHHSN266200600012C from the Department of Health and Human Services(DHHS) and contract number HDTRA-1-07-C-0082, from the Department ofDefense and Defense Threat Reduction Agency (DOD/DTRA). The governmentmay therefore have certain rights in the invention.

BACKGROUND

Oligonucleotide compounds have important therapeutic applications inmedicine. Oligonucleotides can be used to silence genes that areresponsible for a particular disease. Gene-silencing prevents formationof a protein by inhibiting translation. Importantly, gene-silencingagents are a promising alternative to traditional small, organiccompounds that inhibit the function of the protein linked to thedisease. siRNA, antisense RNA, and micro-RNA are oligonucleotides thatprevent the formation of proteins by gene-silencing.

RNA interference or “RNAi” is a term initially coined by Fire andco-workers to describe the observation that double-stranded RNA (dsRNA)can block gene expression when it is introduced into worms (Fire et al.(1998) Nature 391, 806-811). Short dsRNA directs gene-specific,post-transcriptional silencing in many organisms, including vertebrates,and has provided a new tool for studying gene function. RNAi is mediatedby RNA-induced silencing complex (RISC), a sequence-specific,multi-component nuclease that destroys messenger RNAs homologous to thesilencing trigger. RISC is known to contain short RNAs (approximately 22nucleotides) derived from the double-stranded RNA trigger, but theprotein components of this activity remained unknown.

siRNA compounds are promising agents for a variety of diagnostic andtherapeutic purposes. siRNA compounds can be used to identify thefunction of a gene. In addition, siRNA compounds offer enormouspotential as a new type of pharmaceutical agent which acts by silencingdisease-causing genes. Research is currently underway to developinterference RNA therapeutic agents for the treatment of many diseasesincluding central-nervous-system diseases, inflammatory diseases,metabolic disorders, oncology, infectious diseases, and ocular disease.

siRNA has been shown to be extremely effective as a potential anti-viraltherapeutic with numerous published examples appearing recently. siRNAmolecules directed against targets in the viral genome dramaticallyreduce viral titers by orders of magnitude in animal models of influenza(Ge et. al., Proc. Natl. Acd. Sci. USA, 101:8676-8681 (2004); Tompkinset. al., Proc. Natl. Acd. Sci. USA, 101:8682-8686 (2004); Thomas et.al., Expert Opin. Biol. Ther. 5:495-505 (2005)), respiratory synctialvirus (RSV) (Bitko et. al., Nat. Med. 11:50-55 (2005)), hepatitis Bvirus (HBV) (Morrissey et. al., Nat. Biotechnol. 23:1002-1007 (2005)),hepatitis C virus (Kapadia, Proc. Natl. Acad. Sci. USA, 100:2014-2018(2003); Wilson et. al., Proc. Natl. Acad. Sci. USA, 100:2783-2788(2003)) and SARS coronavirus (Li et. al., Nat. Med. 11:944-951 (2005)).

Antisense methodology is the complementary hybridization of relativelyshort oligonucleotides to mRNA or DNA such that the normal, essentialfunctions, such as protein synthesis, of these intracellular nucleicacids are disrupted. Hybridization is the sequence-specific hydrogenbonding via Watson-Crick base pairs of oligonucleotides to RNA orsingle-stranded DNA. Such base pairs are said to be complementary to oneanother.

The naturally-occurring events that alter the expression level of thetarget sequence, discussed by Cohen (Oligonucleotides: AntisenseInhibitors of Gene Expression, CRC Press, Inc., 1989, Boca Raton, Fla.)are thought to be of two types. The first, hybridization arrest,describes the terminating event in which the oligonucleotide inhibitorbinds to the target nucleic acid and thus prevents, by simple sterichindrance, the binding of essential proteins, most often ribosomes, tothe nucleic acid. Methyl phosphonate oligonucleotides (Miller et al.(1987) Anti-Cancer Drug Design, 2:117-128), and α-anomeroligonucleotides are the two most extensively studied antisense agentswhich are thought to disrupt nucleic acid function by hybridizationarrest.

Another means by which antisense oligonucleotides alter the expressionlevel of target sequences is by hybridization to a target mRNA, followedby enzymatic cleavage of the targeted RNA by intracellular RNase H. A2′-deoxyribofuranosyl oligonucleotide or oligonucleotide analoghybridizes with the targeted RNA and this duplex activates the RNase Henzyme to cleave the RNA strand, thus destroying the normal function ofthe RNA. Phosphorothioate oligonucleotides are the most prominentexample of an antisense agent that operates by this type of antisenseterminating event.

The opportunity to use these and other nucleic acid based therapiesholds significant promise, providing solutions to medical problems thatcould not be addressed with current, traditional medicines. The locationand sequences of an increasing number of disease-related genes are beingidentified, and clinical testing of nucleic acid-based therapeutics fora variety of diseases is now underway.

Despite the advances in application of oligonucleotides andoligonucleotide analogs as therapeutics, the need exists foroligonucleotides having improved pharmacologic properties. Efforts aimedat improving the transmembrane delivery of nucleic acids andoligonucleotides have utilized protein carriers, antibody carriers,liposomal delivery systems, electroporation, direct injection, cellfusion, viral vectors, and calcium phosphate-mediated transformation.However, many of these techniques are limited by the types of cells inwhich transmembrane transport is enabled and by the conditions neededfor achieving such transport. Some progress has been made on increasingthe cellular uptake of single-stranded oligonucleotides, includingincreasing the membrane permeability via conjugates and cellulardelivery of oligonucleotides. In U.S. Pat. No. 6,656,730, M. Manoharandescribes compositions in which a ligand that binds serum, vascular, orcellular proteins may be attached via an optional linking moiety to oneor more sites on an oligonucleotide. These sites include one or more of,but are not limited to, the 2′-position, 3′-position, 5′-position, theinternucleotide linkage, and a nucleobase atom of any nucleotideresidue.

Unlike many of the methods mentioned above, receptor mediatedendocytotic activity can be used successfully both in vitro and in vivo.This mechanism of uptake involves the movement of ligands bound tomembrane receptors into the interior of an area that is enveloped by themembrane via invagination of the membrane structure. This process isinitiated via activation of a cell-surface or membrane receptorfollowing binding of a specific ligand to the receptor. Manyreceptor-mediated endocytotic systems are known and have been studied,including those that recognize sugars such as galactose, mannose,mannose-6-phosphate, peptides and proteins such as transferrin,asialoglycoprotein, vitamin B12, insulin and epidermal growth factor(EGF). The Asialoglycoprotein receptor (ASGP-R) is a high capacityreceptor, which is highly abundant on hepatocytes. The ASGP-R shows a50-fold higher affinity for N-Acetyl-D-Galactosylamine (GalNAc) thanD-Gal. Previous work has shown that multivalency is required to achievenM affinity, while spacing among sugars is also crucial. MultivalentGalNAc clusters and galactosylated carrier systems have beensuccessfully used to target small molecules to hepatocytes in vivo andin vitro.

Receptor mediated endocytosis has been well studied and is known to be acritical pathway for the uptake and internalization of a variety ofcellular nutrients. These are highly developed mechanisms because oftheir critical role in providing nutrients to cells and in maintainingcellular physiology. Thus many examples of the utilization of receptormediated endocytosis pathways for the delivery of drugs, proteins,nucleic acids and other molecules to cells are known.

One way in which this has been applied is the conjugation of essentialnutrients that are actively transported into cells with the drug ormolecule of interest. The transporters or receptors involved in theuptake are capable of recognizing the nutrient portion of the conjugateand ferrying the entire conjugate into the cell. Examples of nutrientsthat are actively transported into cells and that may be of use inconjugates include, but are not limited to, folic acid, vitamin B6,cholesterol and vitamin B12. Such molecules have been conjugated tomacromolecules such as nucleic acids and oligonucleotides to affordconjugates that exhibit improved cellular penetration. Manorharan etal., PCT Application WO 93/07883; Low et al., U.S. Pat. Nos. 5,108,921,5,416,016.

Folic acid and its various forms, such as dihydrofolate andtetrahydrofolate, are essential vitamins that are crucial for thebiosynthesis of nucleic acids and therefore are critical to the survivaland proliferation of cells. Folate cofactors play an important role inthe one-carbon transfers that are critical for the biosynthesis ofpyrimidine nucleosides. Cells therefore have a sophisticated system oftransporting folates into the cytoplasm. Uptake of folates occurs by twodifferent pathways depending on the cell type. Cells expressing acarrier or transporter for folate that exhibits a low affinity (Kd˜10⁻⁶M) for the vitamin prefer reduced over oxidized forms of folate. Cellsthat express membrane receptors called folate binding protein (FBP), incontrast, exhibit high binding affinity (Kd˜10⁻⁹ M) and prefer theoxidized form of the vitamin. This latter receptor is believed tomediate the uptake of folates into the cytoplasm via endocytosis.

The use of biotin conjugates and also folic acid conjugates to enhancetransmembrane transport of exogenous molecules, includingoligonucleotides, has been reported by Low et al., U.S. Pat. Nos.5,108,921; 5,416,016; PCT Application WO 90/12096. Folic acid wasconjugated to 3′-aminoalkyl-oligonucleotides at their 3′-terminus viacarbodiimide chemistry. The multiplicity of folate receptors on membranesurfaces of most cells and the associated receptor mediated endocytoticprocesses were implicated in the enhanced transport of theseoligonucleotide-folic acid conjugates into cells. There are however,several limitations to this approach for the conjugation of folic acidto oligonucleotides.

Folic acid and many related folates and antifolates exhibit very poorsolubility that hinders the effective conjugation of folic acid tooligonucleotides and subsequent purification of oligonucleotide-folicacid conjugates. Further folic acid bears two reactive carboxylic acidgroups that are just as likely to react with the terminal amino group ofthe 3-aminoalkyl-oligonucleotide. Thus conjugation will typically resultin a mixture of a- and g-conjugates arising from the reaction of thea-carboxylate and the g-carboxylate of the glutamic acid portion of thefolic acid molecule. This poses difficulties from the standpoint ofcharacterizing the conjugate and further from the standpoint ofpolyglutamylation of folates. Polyglutamylation of folates is a wellrecognized phenomenon that has significant implications on thetransport, localization and activity of folates. Since polyglutamylationrates differ significantly between the α- and γ-carboxylates, the use ofpoorly defined mixtures of oligonucleotide-folate conjugates, asobtained from the Low et al. procedure, U.S. Pat. No. 5,108,921, willlead to variable transport and concentration of the conjugate. Further,the conjugation of folates onto one end of an oligonucleotide may be adisadvantage because of the known propensity of exonucleases to rapidlycleave oligonucleotides by excising the terminal residues. Also, it hasbeen observed that oligonucleotide-folic acid conjugates prepared inthis fashion are light sensitive.

Therefore, there is a clear need for new oligonucleotide-folateconjugates, oligonucleotide-carbohydrate conjugates and methods fortheir preparation, that address the shortcomings of oligonucleotideconjugates as described above. The present invention is directed to thisvery important end.

SUMMARY OF THE INVENTION

The present invention provides compounds of formula I

wherein:

X is H, a hydroxyl protecting group, a phosphate group, a phosphodiestergroup, an activated phosphate group, an activated phosphite group, aphosphoramidite, a solid support, —P(Z′)(Z″)O-nucleoside,—P(Z′)(Z″)O-oligonucleotide, a lipid, a PEG, a steroid, a polymer,—P(Z′)(Z″)O-L⁶-Q′-L⁷-OP(Z′″)(Z″″)O-oligonucleotide, a nucleotide, or anoligonucleotide;

Y is H, a hydroxyl protecting group, a phosphate group, a phosphodiestergroup, an activated phosphate group, an activated phosphite group, aphosphoramidite, a solid support, —P(Z′)(Z″)O-nucleoside,—P(Z′)(Z″)O-oligonucleotide, a lipid, a PEG, a steroid, a lipophile, apolymer, —P(Z′)(Z″)O-L⁶-Q′-L⁷-OP(Z′″)(Z″″)O-oligonucleotide, anucleotide, or an oligonucleotide;

Q is a tether;

R is folate, a folate analog, a folate mimic, a folate receptor bindingligand;

Z′, Z″, Z′″ and Z″″ are independently O or S;

n represent independently for each occurrence 1-20;

m represent independently for each occurrence 0-50;

L⁶ and L⁷ are each independently for each occurrence —(CH₂)_(n)—,—C(R′)(R″)(CH₂)_(n)—, —(CH₂)C(R′)(R″)—, —(CH₂CH₂O)_(m)CH₂CH₂—, or—(CH₂CH₂O)_(m)CH₂CH₂NH—;

Q′ is NH, O, S, CH₂, C(O)O, C(O)NH, —NH—CH(R^(a))—C(O)—,—C(O)—CH(R^(a))—NH—, CO,

R^(a) is H or amino acid side chain.

In some preferred embodiments R is folate, a folate analog, a folatemimic or a folate receptor binding ligand.

In some embodiments, R is

Embodiments can include one or more of the features described above.

In a further aspect, this invention features an iRNA agent having afirst strand and a second strand, wherein at least one subunit having aformula (I) is incorporated into at least one of said strands.

In one aspect, this invention features an iRNA agent having a firststrand and a second strand, wherein at least two subunits having aformula (I) are incorporated into at least one of said strands.

In another aspect, this invention provides a method of making an iRNAagent described herein having a first strand and a second strand inwhich at least one subunit of formula (I) is incorporated in thestrands. The method includes contacting the first strand with the secondstrand.

In a further aspect, this invention provides a method of modulatingexpression of a target gene, the method includes administering an iRNAagent described herein having a first strand and a second strand inwhich at least one subunit of formula (I) is incorporated in the strandsto a subject.

In one aspect, this invention features a pharmaceutical compositionhaving an iRNA agent described herein having a first strand and a secondstrand in which at least one subunit of formula (I) is incorporated inthe strands and a pharmaceutically acceptable carrier.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1. Graphical representation of folate conjugated sequences.

FIG. 2. Graphical representation of sequences AL-3609 and AL-3610.

FIG. 3. Graphical representation of sequences AL-3664, AL-3665, AL-3670and AL-3671.

FIG. 4. Graphical representation of sequences AL-3639, AL-3640, AL-3668and AL-3669.

FIG. 5. Folate conjugate monomer (compound 108) used in the synthesis ofoligonucleotides.

FIG. 6. Cholesterol conjugate monomer used in the synthesis ofoligonucleotides.

FIG. 7. C18 Spacer monomer used in the synthesis of oligonucleotides.

FIG. 8. 12 Disulfide linker monomer used in the synthesis ofoligonucleotides.

FIG. 9. A schematic view of oligonucleotide conjugate designconsiderations.

FIG. 10. Comparison of binding of various folate conjugates to folatereceptor.

FIG. 11. Effect of folate conjugation on the silencing activity ofsiRNAs.

FIG. 12. In vivo targeting of folate conjugated siRNAs.

FIG. 13. Tissue levels of folate conjugated siRNAs in vivo.

FIG. 14. Tumor distribution of folate conjugated siRNAs.

FIG. 15. Co-localization of folate conjugated siRNAs with macrophages.

FIG. 16. Establishment of KB EGFP tumors in nude mice.

FIG. 17. Conjugates with endosomolytic linkers.

DETAILED DESCRIPTION

The inventor has discovered, inter alia, that attachment of a folic acidmoiety to an iRNA agent can optimize one or more properties of the iRNAagent. In many cases, the folic acid will be attached to a modifiedsubunit of the iRNA agent. E.g., the ribose sugar of one or moreribonucleotide subunits of an iRNA agent can be replaced with anothermoiety, e.g., a non-carbohydrate (preferably cyclic) carrier to which isattached a folic acid. A ribonucleotide subunit in which the ribosesugar of the subunit has been so replaced is referred to herein as aribose replacement modification subunit (RRMS). A cyclic carrier may bea carbocyclic ring system, i.e., all ring atoms are carbon atoms, or aheterocyclic ring system, i.e., one or more ring atoms may be aheteroatom, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be amonocyclic ring system, or may contain two or more rings, e.g. fusedrings. The cyclic carrier may be a fully saturated ring system, or itmay contain one or more double bonds.

The carriers further include (i) at least two “backbone attachmentpoints” and (ii) at least one “tethering attachment point.” A “backboneattachment point” as used herein refers to a functional group, e.g. ahydroxyl group, or generally, a bond available for, and that is suitablefor incorporation of the carrier into the backbone, e.g., the phosphate,or modified phosphate, e.g., sulfur containing, backbone, of aribonucleic acid. A “tethering attachment point” in some embodimentsrefers to a constituent ring atom of the cyclic carrier, e.g., a carbonatom or a heteroatom (distinct from an atom which provides a backboneattachment point), that connects a selected moiety. The moiety can be,e.g., a folic acid, folic acid analog, a folic acid mimic or a ligandcapable of binding to the folate receptor. Optionally, the selectedmoiety is connected by an intervening tether to the cyclic carrier.Thus, the cyclic carrier will often include a functional group, e.g., anamino group, or generally, provide a bond, that is suitable forincorporation or tethering of another chemical entity, e.g., a ligand tothe constituent ring.

In addition to the cyclic carriers described herein, RRMS can includecyclic carriers described in copending co-owned U.S. application Ser.No. 10/916,185 filed Aug. 10, 2004, and U.S. application Ser. No.10/946,873 filed Sep. 21, 2004, both of which are hereby incorporated byreference.

Accordingly, in one aspect, the invention features, a monomer having thestructure shown in formula (I′)

wherein:

A and B are each independently for each occurrence O, N(R^(N)) or S;

X is H, a protecting group, a phosphate group, a phosphodiester group,an activated phosphate group, an activated phosphite group, aphosphoramidite, a solid support, —P(Z′)(Z″)O-nucleoside,—P(Z′)(Z″)O-oligonucleotide, a lipid, a PEG, a steroid, a polymer,—P(Z′)(Z″)O-L⁶-Q′-L⁷-OP(Z′″)(Z″″)O-oligonucleotide, a nucleotide, or anoligonucleotide;

Y is H, a protecting group, a phosphate group, a phosphodiester group,an activated phosphate group, an activated phosphite group, aphosphoramidite, a solid support, —P(Z′)(Z″)O-nucleoside,—P(Z′)(Z″)O-oligonucleotide, a lipid, a PEG, a steroid, a lipophile, apolymer, —P(Z′)(Z″)O-L⁶-Q′-L⁷-OP(Z′″)(Z″″)O-oligonucleotide, anucleotide, or an oligonucleotide;

R is folate, a folate analog a folate mimic or a folate receptor bindingligand;

L⁶ and L⁷ are each independently for each occurrence —(CH₂)_(n)—,—C(R′)(R″)(CH₂)_(n)—, —(CH₂)_(n)C(R′)(R″)—, —(CH₂CH₂O)_(m)CH₂CH₂—, or—(CH₂CH₂O)_(m)CH₂CH₂NH—;

Q′ is NH, O, S, CH₂, C(O)O, C(O)NH, —NH—CH(R^(a))—C(O)—,—C(O)—CH(R^(a))—NH—, CO,

R^(a) is H or amino acid side chain;

R′ and R″ are each independently H, CH₃, OH, SH, NH₂, NH(Alkyl=Me, Et,Pr, isoPr, Bu, Bn) or N(diAlkyl=Me₂, Et₂, Bn₂);

Z′, Z″, Z′″ and Z″″ are independently O or S;

n represent independently for each occurrence 1-20; and

m represent independently for each occurrence 0-50.

Accordingly, in one aspect, the invention features, a monomer having thestructure shown in formula (I).

wherein:

X is H, a hydroxyl protecting group, a phosphate group, a phosphodiestergroup, an activated phosphate group, an activated phosphite group, aphosphoramidite, a solid support, —P(Z′)(Z″)O-nucleoside,—P(Z′)(Z″)O-oligonucleotide, a lipid, a PEG, a steroid, a polymer,—P(Z′)(Z″)O-L⁶-Q′-L⁷-OP(Z′″)(Z″″)O-oligonucleotide, a nucleotide, or anoligonucleotide;

Y is H, a hydroxyl protecting group, a phosphate group, a phosphodiestergroup, an activated phosphate group, an activated phosphite group, aphosphoramidite, a solid support, —P(Z′)(Z″)O-nucleoside,—P(Z′)(Z″)O-oligonucleotide, a lipid, a PEG, a steroid, a lipophile, apolymer, —P(Z′)(Z″)O-L⁶-Q′-L⁷-OP(Z′″)(Z″″)O-oligonucleotide, anucleotide, or an oligonucleotide;

Q is a tether;

R is folate, a folate analog, a folate mimic, a folate receptor bindingligand;

n represent independently for each occurrence 1-20;

m represent independently for each occurrence 0-50;

L⁶ and L⁷ are each independently for each occurrence —(CH₂)_(n)—,—C(R′)(R″)(CH₂)_(n)—, —(CH₂)_(n)C(R′)(R″)—, —(CH₂CH₂O)_(m)CH₂CH₂—, or—(CH₂CH₂O)_(m)CH₂CH₂NH—;

Q′ is NH, O, S, CH₂, C(O)O, C(O)NH, —NH—CH(R^(a))—C(O)—,—C(O)—CH(R^(a))—NH—, CO,

R^(a) is H or amino acid side chain.

In some embodiments, R is

In some embodiments, R is one of

In some embodiments, R is one of

In some embodiments, R is one of

In some embodiments, R is

In some embodiments, the RRMS has the structure

In some embodiments, the RRMS has the structure

In some embodiments, RRMS has the structure

In some embodiments, RRMS has the structure

In some embodiments, RRMS has the structure

In some embodiments, RRMS has the structure

In some embodiments, RRMS has the structure

In some embodiments, RRMS has the structure

In some embodiments, RRMS has the

structure

In one aspect, the invention features, a compound having the structureshown in formula (CI)

A and B are independently for each occurrence hydrogen, protectinggroup, optionally substituted aliphatic, optionally substituted aryl,optionally substituted heteroaryl, polyethyleneglycol (PEG), aphosphate, a diphosphate, a triphosphate, a phosphonate, aphosphonothioate, a phosphonodithioate, a phosphorothioate, aphosphorothiolate, a phosphorodithioate, a phosphorothiolothionate, aphosphodiester, a phosphotriester, an activated phosphate group, anactivated phosphite group, a phosphoramidite, a solid support,—P(Z¹)(Z²)—O-nucleoside, or —P(Z¹)(Z²)—O-oligonucleotide; wherein Z¹ andZ² are each independently for each occurrence O, S, N(alkyl) oroptionally substituted alkyl;

J₁ and J₂ are independently O, S, NR^(N), optionally substituted alkyl,OC(O)NH, NHC(O)O, C(O)NH, NHC(O), OC(O), C(O)O, OC(O)O, NHC(O)NH,NHC(S)NH, OC(S)NH, OP(N(R^(P))₂)O, or OP(N(R^(P))₂); and

is cyclic group or acyclic group; preferably, the cyclic group isselected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl,imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl,isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl,quinoxalinyl, pyridazinonyl, tetrahydrofuryl and decalin; preferably,the acyclic group is selected from serinol backbone or diethanolaminebackbone.

In preferred embodiments, ligand is a folate or folate analog. As usedherein, the term “folate” is meant to refer to folate and folatederivatives, including pteroic acid derivatives and analogs. The analogsand derivatives of folic acid suitable for use in the present inventioninclude, but are not limited to, antifolates, dihy-drofloates,tetrahydrofolates, tetrahydrorpterins, folinic acid, pteropolyglutamicacid, 1-deza, 3-deaza, 5-deaza, 8-deaza, 10-deaza, 1,5-deaza, 5,10dideaza, 8,10-dideaza, and 5,8-dideaza folates, antifolates, and pteroicacid derivatives. Additional folate analogs are described in publishedUS publication US2004/0,242,582 (published Dec. 2, 2004).

In one embodiment, the compound is a pyrroline ring system as shown informula (CII)

wherein E is absent or C(O), C(O)O, C(O)NH, C(S), C(S)NH, SO, SO₂, orSO₂NH;

R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, and R¹⁸ are each independently foreach occurrence H, —CH₂OR^(a), or OR^(b),

R^(a) and R^(b) are each independently for each occurrence hydrogen,hydroxyl protecting group, optionally substituted alkyl, optionallysubstituted aryl, optionally substituted cycloalkyl, optionallysubstituted aralkyl, optionally substituted alkenyl, optionallysubstituted heteroaryl, polyethyleneglycol (PEG), a phosphate, adiphosphate, a triphosphate, a phosphonate, a phosphonothioate, aphosphonodithioate, a phosphorothioate, a phosphorothiolate, aphosphorodithioate, a phosphorothiolothionate, a phosphodiester, aphosphotriester, an activated phosphate group, an activated phosphitegroup, a phosphoramidite, a solid support, —P(Z¹)(Z²)—O-nucleoside,—P(Z¹)(Z²)—O-oligonucleotide, —P(Z¹)(O-linker-R^(L))—O-nucleoside, or—P(Z¹)(O-linker-R^(L))—O-oligonucleotide;

R³⁰ is independently for each occurrence -linker-R^(L) or R³¹;

R^(L) is hydrogen or a ligand;

R³¹ is —C(O)CH(N(R³²)₂)(CH₂)_(h)N(R³²)₂;

R³² is independently for each occurrence H, —R^(L), -linker-R^(L) orR³¹;

Z¹ is independently for each occurrence O or S;

Z² is independently for each occurrence O, S, N(alkyl) or optionallysubstituted alkyl; and

h is independently for each occurrence 1-20.

For the pyrroline-based click-carriers, R¹¹ is —CH₂OR^(a) and R³ isOR^(b); or R¹¹ is —CH₂OR^(a) and R⁹ is OR^(b); or R¹¹ is —CH₂OR^(a) andR¹⁷ is OR^(b); or R¹³ is —CH₂OR^(a) and R¹¹ is OR^(b); or R¹³ is—CH₂OR^(a) and R¹⁵ is OR^(b); or R¹³ is —CH₂OR^(a) and R¹⁷ is OR^(b). Incertain embodiments, CH₂OR^(a) and OR^(b) may be geminally substituted.For the 4-hydroxyproline-based carriers, R¹¹ is —CH₂OR^(a) and R¹⁷ isOR^(b). The pyrroline- and 4-hydroxyproline-based compounds maytherefore contain linkages (e.g., carbon-carbon bonds) wherein bondrotation is restricted about that particular linkage, e.g. restrictionresulting from the presence of a ring. Thus, CH₂OR^(a) and OR^(b) may becis or trans with respect to one another in any of the pairingsdelineated above Accordingly, all cis/trans isomers are expresslyincluded. The compounds may also contain one or more asymmetric centersand thus occur as racemates and racemic mixtures, single enantiomers,individual diastereomers and diastereomeric mixtures. All such isomericforms of the compounds are expressly included (e.g., the centers bearingCH₂OR^(a) and OR^(b) can both have the R configuration; or both have theS configuration; or one center can have the R configuration and theother center can have the S configuration and vice versa).

In one embodiment, R¹ is CH₂OR^(a) and R⁹ is OR^(b).

In one embodiment, R^(b) is a solid support.

In one embodiment, carrier of formula (CII) is a phosphoramidite, i.e.,one of R^(a) or R^(b) is —P(O-alkyl)N(alkyl)₂, e.g.,—P(OCH₂CH₂CN)N(i-propyl)₂. In one embodiment, R^(b) is—P(O-alkyl)N(alkyl)₂.

In embodiment, the compound is a ribose ring system as shown in formula(CIII).

wherein:

X is O, S, NR^(N) or CR^(P) ₂;

B is independently for each occurrence hydrogen, optionally substitutednatural or non-natural nucleobase, optionally substituted naturalnucleobase conjugated with -linker-R^(L) or optionally substitutednon-natural nucleobase conjugated with -linker-R^(L);

R¹, R², R³, R⁴ and R⁵ are each independently for each occurrence H, OR⁶,F, N(R^(N))₂, or -J-linker-R_(L);

J is absent, O, S, NR^(N), OC(O)NH, NHC(O)O, C(O)NH, NHC(O), NHSO,NHSO₂, NHSO₂NH, OC(O), C(O)O, OC(O)O, NHC(O)NH, NHC(S)NH, OC(S)NH,OP(N(R^(P))₂)O, or OP(N(R^(P))₂);

R⁶ is independently for each occurrence hydrogen, hydroxyl protectinggroup, optionally substituted alkyl, optionally substituted aryl,optionally substituted cycloalkyl, optionally substituted aralkyl,optionally substituted alkenyl, optionally substituted heteroaryl,polyethyleneglycol (PEG), a phosphate, a diphosphate, a triphosphate, aphosphonate, a phosphonothioate, a phosphonodithioate, aphosphorothioate, a phosphorothiolate, a phosphorodithioate, aphosphorothiolothionate, a phosphodiester, a phosphotriester, anactivated phosphate group, an activated phosphite group, aphosphoramidite, a solid support, —P(Z¹)(Z²)—O-nucleoside,—P(Z¹)(Z²)—O-oligonucleotide, —P(Z¹)(Z²)-formula (CIII),—P(Z¹)(O-linker-R^(L))—O-nucleoside,—P(Z¹)(O-linker-R^(L))—O-oligonucleotide, or—P(Z¹)(O-linker-R^(L))—O-formula (CIII);

R^(N) is independently for each occurrence H, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, optionally substituted cycloalkyl,optionally substituted aralkyl, optionally substituted heteroaryl or anamino protecting group;

R^(P) is independently for each occurrence H, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, optionally substituted cycloalkyl oroptionally substituted heteroaryl;

R^(L) is hydrogen or a ligand;

Z¹ and Z² are each independently for each occurrence O, SN(alkyl) oroptionally substituted alkyl; and

provided that R^(L) is present at least once and further provided thatR^(L) is a ligand at least once.

In one embodiment, the carrier of formula (CI) is an acyclic group andis termed an “acyclic carrier”. Preferred acyclic carriers can have thestructure shown in formula (CIV) or formula (CV) below.

In one embodiment, the compound is an acyclic carrier having thestructure shown in formula (CIV).

wherein:

W is absent, O, S and N(R^(N)), where R^(N) is independently for eachoccurrence H, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substituted aryl,optionally substituted cycloalkyl, optionally substituted aralkyl,optionally substituted heteroaryl or an amino protecting group;

E is absent or C(O), C(O)O, C(O)NH, C(S), C(S)NH, SO, SO₂, or SO₂NH;

R^(a) and R^(b) are each independently for each occurrence hydrogen,hydroxyl protecting group, optionally substituted alkyl, optionallysubstituted aryl, optionally substituted cycloalkyl, optionallysubstituted aralkyl, optionally substituted alkenyl, optionallysubstituted heteroaryl, polyethyleneglycol (PEG), a phosphate, adiphosphate, a triphosphate, a phosphonate, a phosphonothioate, aphosphonodithioate, a phosphorothioate, a phosphorothiolate, aphosphorodithioate, a phosphorothiolothionate, a phosphodiester, aphosphotriester, an activated phosphate group, an activated phosphitegroup, a phosphoramidite, a solid support, —P(Z¹)(Z²)—O-nucleoside,—P(Z¹)(Z²)—O-oligonucleotide, —P(Z¹)(O-linker-R^(L))—O-nucleoside, or—P(Z¹)(O-linker-R^(L))—O-oligonucleotide;

R³⁰ is independently for each occurrence -linker-R^(L) or R³¹;

R^(L) is hydrogen or a ligand;

R³¹ is —C(O)CH(N(R³²)₂)(CH₂)_(h)N(R³²)₂;

R³² is independently for each occurrence H, —R^(L), -linker-R^(L) orR³¹;

Z¹ is independently for each occurrence O or S;

Z² is independently for each occurrence O, S, N(alkyl) or optionallysubstituted alkyl;

h is independently for each occurrence 1-20; and

r, s and t are each independently for each occurrence 0, 1, 2 or 3.

When r and s are different, then the tertiary carbon can be either the Ror S configuration. In preferred embodiments, x and y are one and z iszero (e.g. carrier is based on serinol). The acyclic carriers canoptionally be substituted, e.g. with hydroxy, alkoxy, perhaloalky.

In one embodiment, the compound is an acyclic carrier having thestructure shown in formula (CV)

wherein E is absent or C(O), C(O)O, C(O)NH, C(S), C(S)NH, SO, SO₂, orSO₂NH;

R^(a) and R^(b) are each independently for each occurrence hydrogen,hydroxyl protecting group, optionally substituted alkyl, optionallysubstituted aryl, optionally substituted cycloalkyl, optionallysubstituted aralkyl, optionally substituted alkenyl, optionallysubstituted heteroaryl, polyethyleneglycol (PEG), a phosphate, adiphosphate, a triphosphate, a phosphonate, a phosphonothioate, aphosphonodithioate, a phosphorothioate, a phosphorothiolate, aphosphorodithioate, a phosphorothiolothionate, a phosphodiester, aphosphotriester, an activated phosphate group, an activated phosphitegroup, a phosphoramidite, a solid support, —P(Z¹)(Z²)—O-nucleoside,—P(Z¹)(Z²)—O-oligonucleotide, —P(Z¹)(Z²)-formula (I),—P(Z¹)(O-linker-R^(L))—O-nucleoside, or—P(Z¹)(O-linker-R^(L))—O-oligonucleotide;

R³⁰ is independently for each occurrence -linker-R^(L) or R³¹;

R^(L) is hydrogen or a ligand;

R³¹ is —C(O)CH(N(R³²)₂)(CH₂)_(h)N(R³²)₂;

R³² is independently for each occurrence H, —R^(L), -linker-R^(L) orR³¹;

Z¹ is independently for each occurrence O or S;

Z² is independently for each occurrence O, S, N(alkyl) or optionallysubstituted alkyl; and

h is independently for each occurrence 1-20; and

r and s are each independently for each occurrence 0, 1, 2 or 3.

Other carrier compounds amenable to the invention are described incopending applications U.S. Ser. No. 10/916,185, filed Aug. 10, 2004;U.S. Ser. No. 10/946,873, filed Sep. 21, 2004; U.S. Ser. No. 10/985,426,filed Nov. 9, 2004; U.S. Ser. No. 10/833,934, filed Aug. 3, 2007; U.S.Ser. No. 11/115,989 filed Apr. 27, 2005 and U.S. Ser. No. 11/119,533,filed Apr. 29, 2005, which are incorporated by reference in theirentireties for all purposes.

In some embodiments, ligand is chosen from a group consisting of

Embodiments can include one or more of the features described above.

In a further aspect, this invention features an iRNA agent having afirst strand and a second strand, wherein at least one subunit having aformula (I) is incorporated into at least one of said strands.

In one aspect, this invention features an iRNA agent having a firststrand and a second strand, wherein at least two subunits having aformula (I) are incorporated into at least one of said strands.

In another aspect, this invention provides a method of making an iRNAagent described herein having a first strand and a second strand inwhich at least one subunit of formula (I) is incorporated in thestrands. The method includes contacting the first strand with the secondstrand.

In a further aspect, this invention provides a method of modulatingexpression of a target gene, the method includes administering an iRNAagent described herein having a first strand and a second strand inwhich at least one subunit of formula (I) is incorporated in the strandsto a subject.

In one aspect, this invention features a pharmaceutical compositionhaving an iRNA agent described herein having a first strand and a secondstrand in which at least one subunit of formula (I) is incorporated inthe strands and a pharmaceutically acceptable carrier.

RRMS monomers described herein may be incorporated into anydouble-stranded RNA-like molecule described herein, e.g., an iRNA agent.An iRNA agent may include a duplex comprising a hybridized sense andantisense strand, in which the antisense strand and/or the sense strandmay include one or more of the RRMSs described herein. An RRMS can beintroduced at one or more points in one or both strands of adouble-stranded iRNA agent. An RRMS can be placed at or near (within 1,2, or 3 positions) of the 3′ or 5′ end of the sense strand or at near(within 1, 2 or 3 positions of) the 3′ end of the antisense strand. Insome embodiments it is preferred to not have an RRMS at or near (within1, 2, or 3 positions of) the 5′ end of the antisense strand. An RRMS canbe internal, and will preferably be positioned in regions not criticalfor antisense binding to the target.

In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or3 positions of) the 3′ end of the antisense strand. In an embodiment, aniRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the3′ end of the antisense strand and at (or within 1, 2, or 3 positionsof) the 3′ end of the sense strand. In an embodiment, an iRNA agent mayhave an RRMS at (or within 1, 2, or 3 positions of) the 3′ end of theantisense strand and an RRMS at the 5′ end of the sense strand, in whichboth RRMS are located at the same end of the iRNA agent.

In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or3 positions of) the 3′ end of the antisense strand and an RRMS at the 5′end of the sense strand, in which both RRMSs may share the same ligand(e.g., folic acid) via connection of their individual tethers toseparate positions on the ligand. A ligand shared between two proximalRRMSs is referred to herein as a “hairpin ligand.”

In other embodiments, an iRNA agent may have an RRMS at the 3′ end ofthe sense strand and an RRMS at an internal position of the sensestrand. An iRNA agent may have an RRMS at an internal position of thesense strand; or may have an RRMS at an internal position of theantisense strand; or may have an RRMS at an internal position of thesense strand and an RRMS at an internal position of the antisensestrand.

In preferred embodiments the iRNA agent includes a first and secondsequences, which are preferably two separate molecules as opposed to twosequences located on the same strand, have sufficient complementarity toeach other to hybridize (and thereby form a duplex region), e.g., underphysiological conditions, e.g., under physiological conditions but notin contact with a helicase or other unwinding enzyme.

It is preferred that the first and second sequences be chosen such thatthe double stranded iRNA agent includes a single strand or unpairedregion at one or both ends of the molecule. Thus, a double stranded iRNAagent contains first and second sequences, preferably paired to containan overhang, e.g., one or two 5′ or 3′ overhangs but preferably a 3′overhang of 2-3 nucleotides. Most embodiments will have a 3′ overhang.Preferred iRNA agents will have single-stranded overhangs, preferably 3′overhangs, of 1 or preferably 2 or 3 nucleotides in length at each end.The overhangs can be the result of one strand being longer than theother, or the result of two strands of the same length being staggered.5′ ends are preferably phosphorylated.

Other modifications to sugars, bases, or backbones can be incorporatedinto the iRNA agents.

The iRNA agents can take an architecture or structure described herein.The iRNA agents can be palindromic, or double targeting, as describedherein.

The iRNA agents can have a sequence such that a non-cannonical or otherthan cannonical Watson-Crick structure is formed between two monomers ofthe iRNA agent or between a strand of the iRNA agent and anothersequence, e.g., a target or off-target sequence, as is described herein.

The iRNA agent can be selected to target any of a broad spectrum ofgenes, including any of the genes described herein.

In a preferred embodiment the iRNA agent has an architecture(architecture refers to one or more of overall length, length of aduplex region, the presence, number, location, or length of overhangs,single strand versus double strand form) described herein. E.g., theiRNA agent can be less than 30 nucleotides in length, e.g., 21-23nucleotides. Preferably, the iRNA is 21 nucleotides in length and thereis a duplex region of about 19 pairs. In one embodiment, the iRNA is 21nucleotides in length, and the duplex region of the iRNA is 19nucleotides. In another embodiment, the iRNA is greater than 30nucleotides in length.

In some embodiment the duplex region of the iRNA agent will have,mismatches. Preferably it will have no more than 1, 2, 3, 4, or 5 bases,which do not form canonical Watson-Crick pairs or which do nothybridize. Overhangs are discussed in detail elsewhere herein but arepreferably about 2 nucleotides in length. The overhangs can becomplementary to the gene sequences being targeted or can be othersequence. TT is a preferred overhang sequence. The first and second iRNAagent sequences can also be joined, e.g., by additional bases to form ahairpin, or by other non-base linkers.

In addition to the RRMS-containing bases the iRNA agents describedherein can include nuclease resistant monomers (NRMs) described incopending co-owned U.S. Provisional application Ser. No. 10/553,659filed on Apr. 14, 2006 and International Application No. PCT/US04/07070,both of which are hereby incorporated by reference.

In some embodiments, the iRNA agent will have a monomer with thestructure shown in formula (LI) in addition to monomer of formula (I) orformula (I′).

wherein X⁶ and Y⁶ are each independently H, a hydroxyl protecting group,a phosphate group, a phosphodiester group, an activated phosphate group,an activated phosphite group, a phosphoramidite, a solid support,—P(Z′)(Z″)O-nucleoside, —P(Z′)(Z″)O-oligonucleotide, a lipid, a PEG, asteroid, a polymer, —P(Z′)(Z″)O—R¹-Q′-R²—OP(Z′″)(Z″″)O-oligonucleotide,a nucleotide, or an oligonucleotide, —P(Z′)(Z″)-formula (I) or—P(Z′)(Z″)—;

Q⁶ is absent or —(P⁶-Q⁶-R⁶)_(v)-T⁶-;

P⁶ and T⁶ are each independently for each occurrence absent, CO, NH, O,S, OC(O), NHC(O), CH₂, CH₂NH or CH₂O;

Q⁶ is independently for each occurrence absent, substituted alkylenewherein one or more methylenes can be interrupted or terminated by oneor more of O, S, S(O), SO₂, N(R^(N)), C(R′)═C(R′), C≡C or C(O);

R⁶ is independently for each occurrence absent, NH, O, S, CH₂, C(O)O,C(O)NH, NHCH(R^(a))C(O), —C(O)—CH(R^(a))—NH—, CO, CH═N—O,

or heterocyclyl;

R′ and R″ are each independently H, C₁-C₆ alkyl OH, SH, N(R^(N))₂;

R^(N) is independently for each occurrence hydrogen, methyl, ethyl,propyl, isopropyl, butyl or benzyl;

R^(a) is H or amino acid side chain;

Z′, Z″, Z′″ and Z″″ are each independently for each occurrence O or S;

v represent independently for each occurrence 0-20;

R^(L) is a lipophile (e.g., cholesterol, cholic acid, adamantane aceticacid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,bomeol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine), a vitamin (e.g., folate, vitamin A,biotin, pyridoxal), a peptide, a carbohydrate (e.g., monosaccharide,disaccharide, trisaccharide, tetrasaccharide, oligosaccharide,polysaccharide), an endosomolytic component, a steroid (e.g., uvaol,hecigenin, diosgenin), a terpene (e.g., triterpene, e.g.,sarsasapogenin, Friedelin, epifriedelanol derivatized lithocholic acid),or a cationic lipid.

In some embodiments, one or more, e.g., 1, 2, 3, 4 or 5, monomers offormula (LI) in addition to one or more, e.g. 1, 2, 3, 4, or 5, monomersof formula (I) or formula (I′) are present in the iRNA agent.

In some preferred embodiments only 1 monomer of formula (I) or formula(I′) and 1 monomer of formula (LI) are present in the iRNA agent.

In some embodiments, R^(L) is cholesterol.

In some embodiments, R^(L) is lithocholic.

In some embodiments, R^(L) is oleyl lithocholic.

In a preferred embodiment, the iRNA agent targets an exogenous gene of agenetically modified cell. An exogenous gene can be, for example, aviral or bacterial gene that derives from an organism that has invadedor infected the cell, or the exogenous gene can be any gene introducedinto the cell by natural or artificial means, such as by a geneticrecombination event. An iRNA agent can target a viral gene, for example,such as a hepatitis viral gene (e.g., a gene of an HAV, HBV, or HCV).Alternatively, or in addition, the iRNA agent can silence a reportergene, such as GFP or beta galatosidase and the like. These iRNA agentscan be used to silence exogenous genes in an adherent tumor cell line.

In another aspect, the invention provides, methods of silencing a targetgene by providing an iRNA agent to which a folic acid, folic acid analogor folic acid mimic is conjugated. In a preferred embodiment theconjugated iRNA agent can be used to silence a target gene in anorganism, e.g., a mammal, e.g., a human, or to silence a target gene ina cell line or in cells which are outside an organism. In the case of awhole organism, the method can be used to silence a gene, e.g., a genedescribed herein, and treat a condition mediated by the gene. In thecase of use on a cell which is not part of an organism, e.g., a primarycell line, secondary cell line, tumor cell line, or transformed orimmortalized cell line, the iRNA agent to which a folic acid, folic acidanalog or folic acid mimic is conjugated can be used to silence a gene,e.g., one described herein. Cells which are not part of a whole organismcan be used in an initial screen to determine if an iRNA agent iseffective in silencing a gene. A test in cells which are not part of awhole organism can be followed by testing the iRNA agent in a wholeanimal. In preferred embodiments, the iRNA agent which is conjugated toa a carbohydrate, a steroid, or a steroid tethered to at least onecarbohydrate is conjugated is administered to an organism, or contactedwith a cell which is not part of an organism, in the absence of (or in areduced amount of) other reagents that facilitate or enhance delivery,e.g., a compound which enhances transit through the cell membrane. (Areduced amount can be an amount of such reagent which is reduced incomparison to what would be needed to get an equal amount ofnonconjugated iRNA agent into the target cell).

In a preferred embodiment the iRNA agent is suitable for delivery to acell in vivo, e.g., to a cell in an organism. In another aspect, theiRNA agent is suitable for delivery to a cell in vitro, e.g., to a cellin a cell line.

An iRNA agent to which a folic acid, folic acid analog or folic acidmimic is attached can target any gene described herein and can bedelivered to any cell type described herein, e.g., a cell type in anorganism, tissue, or cell line. Delivery of the iRNA agent can be invivo, e.g., to a cell in an organism, or in vitro, e.g., to a cell in acell line.

In another aspect, the invention provides compositions of iRNA agentsdescribed herein, and in particular compositions of an iRNA agent towhich a folate receptor binding ligand e.g. a folic acid, folic acidanalog or folic acid mimic is conjugated, e.g., a lipophilic conjugatediRNA agent described herein. In a preferred embodiment the compositionis a pharmaceutically acceptable composition.

In preferred embodiments, the composition, e.g., pharmaceuticallyacceptable composition, is free of, has a reduced amount of, or isessentially free of other reagents that facilitate or enhance delivery,e.g., compounds which enhance transit through the cell membrane. (Areduced amount can be an amount of such reagent which is reduced incomparison to what would be needed to get an equal amount ofnonconjugated iRNA agent into the target cell). E.g., the composition isfree of, has a reduced amount of, or is essentially free of: anadditional lipophilic moiety; a transfection agent, e.g., concentrationsof an ion or other substance which substantially alters cellpermeability to an iRNA agent; a transfecting agent such asLipofectamine™ (Invitrogen, Carlsbad, Calif.), Lipofectamine 2000™,TransIT-TKO™ (Mirus, Madison, Wis.), FuGENE 6 (Roche, Indianapolis,Ind.), polyethylenimine, X-tremeGENE Q2 (Roche, Indianapolis, Ind.),DOTAP, DOSPER, Metafectene™ (Biontex, Munich, Germany), and the like.

In a preferred embodiment the composition is suitable for delivery to acell in vivo, e.g., to a cell in an organism. In another aspect, theiRNA agent is suitable for delivery to a cell in vitro, e.g., to a cellin a cell line.

The RRMS-containing iRNA agents can be used in any of the methodsdescribed herein, e.g., to target any of the genes described herein orto treat any of the disorders described herein. They can be incorporatedinto any of the formulations, modes of delivery, delivery modalities,kits or preparations, e.g., pharmaceutical preparations, describedherein. E.g, a kit which includes one or more of the iRNA aentsdescribed herein, a sterile container in which the iRNA agent isdisclosed, and instructions for use.

The methods and compositions of the invention, e.g., the RRSM-containingiRNA agents described herein, can be used with any of the iRNA agentsdescribed herein. In addition, the methods and compositions of theinvention can be used for the treatment of any disease or disorderdescribed herein, and for the treatment of any subject, e.g., anyanimal, any mammal, such as any human.

The methods and compositions of the invention, e.g., the RRMS-containingiRNA agents described herein, can be used with any dosage and/orformulation described herein, as well as with any route ofadministration described herein.

DEFINITIONS

The term “halo” refers to any radical of fluorine, chlorine, bromine oriodine.

The term “alkyl” refers to a hydrocarbon chain that may be a straightchain or branched chain, containing the indicated number of carbonatoms. For example, C₁-C₁₂ alkyl indicates that the group may have from1 to 12 (inclusive) carbon atoms in it. The term “haloalkyl” refers toan alkyl in which one or more hydrogen atoms are replaced by halo, andincludes alkyl moieties in which all hydrogens have been replaced byhalo (e.g., perfluoroalkyl). Alkyl and haloalkyl groups may beoptionally inserted with O, N, or S. The terms “aralkyl” refers to analkyl moiety in which an alkyl hydrogen atom is replaced by an arylgroup. Aralkyl includes groups in which more than one hydrogen atom hasbeen replaced by an aryl group. Examples of “aralkyl” include benzyl,9-fluorenyl, benzhydryl, and trityl groups.

The term “alkenyl” refers to a straight or branched hydrocarbon chaincontaining 2-8 carbon atoms and characterized in having one or moredouble bonds. Examples of a typical alkenyl include, but not limited to,allyl, propenyl, 2-butenyl, 3-hexenyl and 3-octenyl groups. The term“alkynyl” refers to a straight or branched hydrocarbon chain containing2-8 carbon atoms and characterized in having one or more triple bonds.Some examples of a typical alkynyl are ethynyl, 2-propynyl, and3-methylbutynyl, and propargyl. The sp² and sp³ carbons may optionallyserve as the point of attachment of the alkenyl and alkynyl groups,respectively.

The terms “alkylamino” and “dialkylamino” refer to —NH(alkyl) and —N(alkyl)₂ radicals respectively. The term “aralkylamino” refers to a—NH(aralkyl) radical. The term “alkoxy” refers to an —O-alkyl radical,and the terms “cycloalkoxy” and “aralkoxy” refer to an —O-cycloalkyl andO-aralkyl radicals respectively. The term “siloxy” refers to a R₃SiO—radical. The term “mercapto” refers to an SH radical. The term“thioalkoxy” refers to an —S-alkyl radical.

The term “alkylene” refers to a divalent alkyl (i.e., —R—), e.g., —CH₂—,—CH₂CH₂—, and —CH₂CH₂CH₂—. The term “alkylenedioxo” refers to a divalentspecies of the structure —O—R—O—, in which R represents an alkylene.

The term “aryl” refers to an aromatic monocyclic, bicyclic, or tricyclichydrocarbon ring system, wherein any ring atom can be substituted.Examples of aryl moieties include, but are not limited to, phenyl,naphthyl, anthracenyl, and pyrenyl.

The term “cycloalkyl” as employed herein includes saturated cyclic,bicyclic, tricyclic, or polycyclic hydrocarbon groups having 3 to 12carbons, wherein any ring atom can be substituted. The cycloalkyl groupsherein described may also contain fused rings. Fused rings are ringsthat share a common carbon-carbon bond or a common carbon atom (e.g.,spiro-fused rings). Examples of cycloalkyl moieties include, but are notlimited to, cyclohexyl, adamantyl, and norbornyl.

The term “heterocyclyl” refers to a nonaromatic 3-10 memberedmonocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ringsystem having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms ifbicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selectedfrom O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms ofN, O, or S if monocyclic, bicyclic, or tricyclic, respectively), whereinany ring atom can be substituted. The heterocyclyl groups hereindescribed may also contain fused rings. Fused rings are rings that sharea common carbon-carbon bond or a common carbon atom (e.g., spiro-fusedrings). Examples of heterocyclyl include, but are not limited totetrahydrofuranyl, tetrahydropyranyl, piperidinyl, morpholino,pyrrolinyl and pyrrolidinyl.

The term “cycloalkenyl” as employed herein includes partiallyunsaturated, nonaromatic, cyclic, bicyclic, tricyclic, or polycyclichydrocarbon groups having 5 to 12 carbons, preferably 5 to 8 carbons,wherein any ring atom can be substituted. The cycloalkenyl groups hereindescribed may also contain fused rings. Fused rings are rings that sharea common carbon-carbon bond or a common carbon atom (e.g., spiro-fusedrings). Examples of cycloalkenyl moieties include, but are not limitedto cyclohexenyl, cyclohexadienyl, or norbornenyl.

The term “heterocycloalkenyl” refers to a partially saturated,nonaromatic 5-10 membered monocyclic, 8-12 membered bicyclic, or 11-14membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, saidheteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6,or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic,respectively), wherein any ring atom can be substituted. Theheterocycloalkenyl groups herein described may also contain fused rings.Fused rings are rings that share a common carbon-carbon bond or a commoncarbon atom (e.g., spiro-fused rings). Examples of heterocycloalkenylinclude but are not limited to tetrahydropyridyl and dihydropyran.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein any ring atomcan be substituted. The heteroaryl groups herein described may alsocontain fused rings that share a common carbon-carbon bond.

The term “oxo” refers to an oxygen atom, which forms a carbonyl whenattached to carbon, an N-oxide when attached to nitrogen, and asulfoxide or sulfone when attached to sulfur.

The term “acyl” refers to an alkylcarbonyl, cycloalkylcarbonyl,arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent,any of which may be further substituted by substituents.

The term “substituents” refers to a group “substituted” on an alkyl,cycloalkyl, alkenyl, alkynyl, heterocyclyl, heterocycloalkenyl,cycloalkenyl, aryl, or heteroaryl group at any atom of that group.Suitable substituents include, without limitation, alkyl, alkenyl,alkynyl, alkoxy, halo, hydroxy, cyano, nitro, amino, SO₃H, sulfate,phosphate, perfluoroalkyl, perfluoroalkoxy, methylenedioxy,ethylenedioxy, carboxyl, oxo, thioxo, imino (alkyl, aryl, aralkyl),S(O)_(n)alkyl (where n is 0-2), S(O)_(n) aryl (where n is 0-2), S(O)_(n)heteroaryl (where n is 0-2), S(O)_(n) heterocyclyl (where n is 0-2),amine (mono-, di-, alkyl, cycloalkyl, aralkyl, heteroaralkyl, andcombinations thereof), ester (alkyl, aralkyl, heteroaralkyl), amide(mono-, di-, alkyl, aralkyl, heteroaralkyl, and combinations thereof),sulfonamide (mono-, di-, alkyl, aralkyl, heteroaralkyl, and combinationsthereof), unsubstituted aryl, unsubstituted heteroaryl, unsubstitutedheterocyclyl, and unsubstituted cycloalkyl. In one aspect, thesubstituents on a group are independently any one single, or any subsetof the aforementioned substituents.

The terms “adeninyl, cytosinyl, guaninyl, thyminyl, and uracilyl” andthe like refer to radicals of adenine, cytosine, guanine, thymine, anduracil.

A “protected” moiety refers to a reactive functional group, e.g., ahydroxyl group or an amino group, or a class of molecules, e.g., sugars,having one or more functional groups, in which the reactivity of thefunctional group is temporarily blocked by the presence of an attachedprotecting group. Protecting groups useful for the monomers and methodsdescribed herein can be found, e.g., in Greene, T. W., Protective Groupsin Organic Synthesis (John Wiley and Sons: New York), 1981, which ishereby incorporated by reference.

Ligand-Conjugated Monomers

Cyclic sugar replacement-based monomers, e.g., sugar replacement-basedligand-conjugated monomers, are also referred to herein as ribosereplacement monomer subunit (RRMS) monomer compounds. Preferred carriershave the general formula (I) provided below. The carriers are an entitywhich can be incorporated into a strand. Thus, it is understood that thestructures also encompass the situations wherein one (in the case of aterminal position) or two (in the case of an internal position) of theattachment points, e.g., either X or Y in case of terminal position andboth X and Y in case of internal position, is connected to thephosphate, or modified phosphate, e.g., sulfur containing, backbone.

Tethers

In certain embodiments, a moiety, e.g., a ligand may be connectedindirectly to the carrier via the intermediacy of an intervening tether.Tethers are connected to the carrier at a tethering attachment point(TAP) and may include any C₁-C₁₀₀ carbon-containing moiety, (e.g.C₁-C₇₅, C₁-C₅₀, C₁-C₂₀, C₁-C₁₀; C₁, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, orC₁₀), preferably having at least one nitrogen atom. In preferredembodiments, the nitrogen atom forms part of a terminal amino or amido(NHC(O)—) group on the tether, which may serve as a connection point forthe ligand. Preferred tethers (underlined) include TAP-(CH₂—NH—;TAP-C(O)(CH₂)NH—; TAP-NR″″(CH—)_(n)NH—, TAP-C(O)—(CH₂)_(n)—C(O)—;TAP-C(O)—(CH₂)_(n)—C(O)O—; TAP-C(O)—O—; TAP-C(O)—(CH₂)_(n)—NH—C(O)—;TAP-C(O)—(CH₂)_(n) ; TAP-C(O)—NH—; TAP-C(O)—; TAP-(CH₂)_(n)—C(O)—;TAP-(CH₂)_(n)—C(O)O—; TAP-(CH₂)_(n) ; or TAP-(CH₂)_(n)—NH—C(O)—;TAP-C(O)—(CH₂)_(n)—NH—C(O)—(CH₂)_(n)CH(R′″)NH—;TAP-C(O)—(CH₂)_(n)—NH—C(O)—(CH₂)_(n)—C(R′)(R″)—SS—(CH₂)_(n)—NH—C(O)—(CH₂)_(n)CH(R′″)NH—;TAP-C(O)—(CH₂)_(n)—NH—C(O)—(CH₂)_(n)—SS—(CH₂)_(n)CH(R′″)—NH—C(O)—(CH₂)_(n)CH(R′″)NH—;TAP-(CH₂)_(n)—NH—C(O)—(CH₂)_(n)C(R′)(R″)—SS—(CH₂)_(n)—; in which each nis independently 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20), R′, R″ and R′″ are described elsewhereand R″″ is C₁-C₆ alkyl. Preferably, n is 2, 5, 6, or 11. In otherembodiments, the nitrogen may form part of a terminal oxyamino group,e.g., —ONH₂, or hydrazino group, —NHNH₂. The tether may optionally besubstituted, e.g., with hydroxy, alkoxy, perhaloalkyl, and/or optionallyinserted with one or more additional heteroatoms, e.g., N, O, or S.Preferred tethered ligands may include, e.g., TAP-(CH₂)_(n)NH(LIGAND);TAP-C(O)(CH₂)_(n)NH(LIGAND); TAP-NR″″(CH₂)_(n)NH(LIGAND);TAP-(CH₂)_(n)ONH(LIGAND); TAP-C(O)(CH₂)_(n)ONH(LIGAND);TAP-NR″″(CH₂)_(n)ONH(LIGAND); TAP-(CH₂)_(n)NHNH₂(LIGAND),TAP-C(O)(CH₂)_(n)NHNH₂(LIGAND); TAP-NR″″(CH₂)_(n)NHNH₂(LIGAND);TAP-C(O)—(CH₂)_(n)—C(O)(LIGAND); TAP-C(O)—(CH₂)_(n)—C(O)O(LIGAND);TAP-C(O)—O(LIGAND); TAP-C(O)—(CH₂)_(n)—NH—C(O)(LIGAND);TAP-C(O)—(CH₂)_(n)(LIGAND); TAP-C(O)—NH(LIGAND); TAP-C(O)(LIGAND);TAP-(CH₂)_(n)—C(O) (LIGAND); TAP-(CH₂)—C(O)O(LIGAND);TAP-(CH₂)_(n)(LIGAND); TAP-(CH₂)_(n)—NH—C(O)(LIGAND);TAP-C(O)—(CH₂)_(n)—NH—C(O)—(CH₂)_(n)CH(R′″)NH(LIGAND);TAP-C(O)—(CH₂)_(n)—NH—C(O)—(CH₂)_(n)C(R′)(R′)—SS—(CH₂)_(n)—NH—C(O)—(CH₂)_(n)CH(R′″)NH(LIGAND);TAP-C(O)—(CH₂)_(n)—NH—C(O)—(CH₂)_(n)—SS—(CH₂)_(n)CH(R′″)—NH—C(O)—(CH₂)_(n)CH(R′″)NH(LIGAND);TAP-(CH₂)_(n)—NH—C(O)—(CH₂)_(n)C(R′)(R″)—SS—(CH₂)_(n)(LIGAND). In someembodiments, amino terminated tethers (e.g., NH₂, ONH₂, NH₂NH₂) can forman imino bond (i.e., C═N) with the ligand. In some embodiments, aminoterminated tethers (e.g., NH₂, ONH₂, NH₂NH₂) can be acylated, e.g., withC(O)CF₃.

In some embodiments, the tether can terminate with a mercapto group(i.e., SH) or an olefin (e.g., CH═CH₂). For example, the tether can beTAP-(CH₂)_(n)—SH, TAP-C(O)(CH₂)_(n)SH, TAP-(CH₂)_(n)—(CH═CH₂₁—, orTAP-C(O)(CH₂)_(n)(CH═CH₂—, in which n can be as described elsewhere. Thetether may optionally be substituted, e.g., with hydroxy, alkoxy,perhaloalkyl, and/or optionally inserted with one or more additionalheteroatoms, e.g., N, O, or S. The double bond can be cis or trans or Eor Z.

In other embodiments the tether may include an electrophilic moiety,preferably at the terminal position of the tether. Preferredelectrophilic moieties include, e.g., an aldehyde, alkyl halide,mesylate, tosylate, nosylate, or brosylate, or an activated carboxylicacid ester, e.g. an NHS ester, or a pentafluorophenyl ester. Preferredtethers (underlined) include TAP-(CH₂)_(n)CHO; TAP-C(O)(CH₂)_(n)CHO; orTAP-NR″″(CH₂)_(n)CHO, in which n is 1-6 and R″″ is C₁-C₆ alkyl; orTAP-(CH₂)_(n)C(O)ONHS; TAP-C(O)(CH₂)_(n)C(O)ONHS; orTAP-NR″″(CH₂)_(n)C(O)ONHS, in which n is 1-6 and R″″ is C₁-C₆ alkyl;TAP-(CH₂)_(n)C(O)OC₆F₅ ; TAP-C(O)(CH₂)_(n)C(O)OC₆F₅ ; orTAP-NR″″(CH₂)_(n)C(O)OC₆F₅ , in which n is 1-11 and R″″ is C₁-C₆ alkyl;or —(CH₂)_(n)CH₂LG; TAP-C(O)(CH₂)_(n)CH₂LG; or TAP-NR″″(CH₂)_(n)CH₂LG,in which n can be as described elsewhere and R″″ is C₁-C₆ alkyl (LG canbe a leaving group, e.g., halide, mesylate, tosylate, nosylate,brosylate). Tethering can be carried out by coupling a nucleophilicgroup of a ligand, e.g., a thiol or amino group with an electrophilicgroup on the tether.

In other embodiments, it can be desirable for the monomer to include aphthalimido group (K) at the terminal position of the tether.

In other embodiments, other protected amino groups can be at theterminal position of the tether, e.g., alloc, monomethoxy trityl (MMT),trifluoroacetyl, Fmoc, or aryl sulfonyl (e.g., the aryl portion can beortho-nitrophenyl or ortho, para-dinitrophenyl).

Any of the tethers described herein may further include one or moreadditional linking groups, e.g., —O—(CH₂)_(n)—, —(CH₂)_(n)—SS—, —(CH₂)—,or —(CH═CH)—.

Ligands

A wide variety of entities, e.g., ligands, can be tethered to an iRNAagent, e.g., to the carrier of a ligand-conjugated monomer subunit.Examples are described below in the context of a ligand-conjugatedmonomer subunit but that is only preferred, entities can be coupled atother points to an iRNA agent.

Preferred moieties are ligands, which are coupled, preferablycovalently, either directly or indirectly via an intervening tether, tothe carrier. In preferred embodiments, the ligand is attached to thecarrier via an intervening tether. As discussed above, the ligand ortethered ligand may be present on the ligand-conjugated monomer when theligand-conjugated monomer is incorporated into the growing strand. Insome embodiments, the ligand may be incorporated into a “precursor”ligand-conjugated monomer subunit after a “precursor” ligand-conjugatedmonomer subunit has been incorporated into the growing strand. Forexample, a monomer having, e.g., an amino-terminated tether, e.g.,TAP-(CH₂)NH₂ may be incorporated into a growing sense or antisensestrand. In a subsequent operation, i.e., after incorporation of theprecursor monomer subunit into the strand, a ligand having anelectrophilic group, e.g., a pentafluorophenyl ester or aldehyde group,can subsequently be attached to the precursor ligand-conjugated monomerby coupling the electrophilic group of the ligand with the terminalnucleophilic group of the precursor ligand-conjugated monomer subunittether.

In preferred embodiments, a ligand alters the distribution, targeting orlifetime of an iRNA agent into which it is incorporated. In preferredembodiments a ligand provides an enhanced affinity for a selectedtarget, e.g, molecule, cell or cell type, compartment, e.g., a cellularor organ compartment, tissue, organ or region of the body, as, e.g.,compared to a species absent such a ligand.

Preferred ligands can improve transport, hybridization, and specificityproperties and may also improve nuclease resistance of the resultantnatural or modified oligoribonucleotide, or a polymeric moleculecomprising any combination of monomers described herein and/or naturalor modified ribonucleotides.

Ligands in general can include therapeutic modifiers, e.g., forenhancing uptake; diagnostic compounds or reporter groups e.g., formonitoring distribution; cross-linking agents; nuclease-resistanceconferring moieties; and natural or unusual nucleobases.

General examples include lipids, steroids (e.g., cholesterol, uvaol,hecigenin, diosgenin), terpenes (e.g., triterpenes, e.g.,sarsasapogenin, Friedelin, epifriedelanol derivatized lithocholic acid),vitamins (e.g., folic acid, folic acid mimics, folate receptor bindingligands, vitamin A, vitamin E, biotin, pyridoxal), carbohydrates,proteins, protein binding agents, integrin targeting molecules, CCR5receptor antagonists, CCR5 receptor binding ligands, polycationics(e.g., porphyrins), peptides, polyamines, peptide mimics, PEG. In someembodiments, the ligand can be one of the following:

Some ligands can have endosomolytic properties. The endosomolyticligands promote the lysis of the endosome and/or transport of thecomposition of the invention, or its components, from the endosome tothe cytoplasm of the cell. The endosomolytic ligand may be a polyanionicpeptide or peptidomimetic which shows pH-dependent membrane activity andfusogenicity. In certain embodiments, the endosomolytic ligand assumesits active conformation at endosomal pH. The “active” conformation isthat conformation in which the endosomolytic ligand promotes lysis ofthe endosome and/or transport of the composition of the invention, orits components, from the endosome to the cytoplasm of the cell.Exemplary endosomolytic ligands include the GAL4 peptide (Subbarao etal., Biochemistry, 1987, 26: 2964-2972), the EALA (SEQ ID NO: 1) peptide(Vogel et al., J. Am. Chem. Soc., 1996, 118: 1581-1586), and theirderivatives (Turk et al., Biochem. Biophys. Acta, 2002, 1559: 56-68). Incertain embodiments, the endosomolytic component may contain a chemicalgroup (e.g., an amino acid) which will undergo a change in charge orprotonation in response to a change in pH. The endosomolytic componentmay be linear or branched. Exemplary primary sequences of peptide basedendosomolytic ligands are shown in Table 1.

TABLE 1 List of peptides with endosomolytic activity. SEQ ID NameSequence (N to C) Ref. NO: GALA AALEALAEALEALAEALEALAEAAAAGGC 1 2 EALAAALAEALAEALAEALAEALAEALAAAAGGC 2 3 (SEQ ID NO: 1) ALEALAEALEALAEA 3 4INF-7 GLFEAIEGFIENGWEGMIWDYG 4 5 Inf  GLFGAIAGFIENGWEGMIDGWYG 5 6 HA-2diINF- GLF EAI EGFI ENGW EGMI DGWYGC 5 7 7 GLF EAI EGFI ENGW EGMI DGWYGCdiINF3 GLF EAI EGFI ENGW EGMI DGGC 6 8 GLF EAI EGFI ENGW EGMI DGGC GLFGLFGALAEALAEALAEHLAEALAEALEALAAGGS 6 9 C GALA-GLFEAIEGFIENGWEGLAEALAEALEALAAGGSC 6 10 INF3 INF-5GLF EAI EGFI ENGW EGnI DG K 4 11 GLF EAI EGFI ENGW EGnI DG 12 n,norleucine References 1. Subbarao et al., Biochemistry, 1987, 26:2964-2972. 2. Vogel et al., J. Am. Chem. Soc., 1996, 118: 1581-1586 3.Turk, M. J., Reddy, J. A. et al. (2002). Characterization of a novelpH-sensitive peptide that enhances drug release from folate-targetedliposomes at endosomal pHs. Biochim. Biophys. Acta 1559, 56-68. 4.Plank, C. Oberhauser, B. Mechtler, K. Koch, C. Wagner, E. (1994). Theinfluence of endosome-disruptive peptides on gene transfer usingsynthetic virus-like gene transfer systems, J. Biol. Chem. 26912918-12924. 5. Mastrobattista, E., Koning, G. A. et al. (2002).Functional characterization of an endosome-disruptive peptide and itsapplication in cytosolic delivery of immunoliposome-entrapped proteins.J. Biol. Chem. 277, 27135-43. 6. Oberhauser, B., Plank, C. et al.(1995). Enhancing endosomal exit of nucleic acids using pH-sensitiveviral fusion peptides. Deliv. Strategies Antisense Oligonucleotide Ther.247-66.

Ligands can improve transport, hybridization, and specificity propertiesand may also improve nuclease resistance of the resultant natural ormodified oligoribonucleotide, or a polymeric molecule comprising anycombination of monomers described herein and/or natural or modifiedribonucleotides.

Ligands in general can include therapeutic modifiers, e.g., forenhancing uptake; diagnostic compounds or reporter groups e.g., formonitoring distribution; cross-linking agents; and nuclease-resistanceconferring moieties. General examples include lipids, steroids,vitamins, sugars, proteins, peptides, polyamines, and peptide mimics.

Ligands can include a naturally occurring substance, such as a protein(e.g., human serum albumin (HSA), low-density lipoprotein (LDL),high-density lipoprotein (HDL), or globulin); an carbohydrate (e.g., adextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronicacid); or a lipid. The ligand may also be a recombinant or syntheticmolecule, such as a synthetic polymer, e.g., a synthetic polyamino acid,an oligonucleotide (e.g. an aptamer). Examples of polyamino acidsinclude polyamino acid is a polylysine (PLL), poly L-aspartic acid, polyL-glutamic acid, styrene-maleic acid anhydride copolymer,poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydridecopolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane,poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, orpolyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic lipid, cationic porphyrin,quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucosamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, biotin, an RGD peptide, an RGDpeptide mimetic or an aptamer. Table 2 shows some examples of targetingligands and their associated receptors.

TABLE 2 Targeting Ligands and their associated receptors Liver CellsLigand Receptor Negatively charged Scavenger receptors molecules 1)Parenchymal Cell (PC) Galactose ASGP-R (Hepatocytes) (Asiologlycoproteinreceptor) Gal NAc ASPG-R (n-acetyl-galactosamine) Gal NAc ReceptorLactose Asialofetuin ASPG-r 2) Sinusoidal Endothelial HyaluronanHyaluronan receptor Cell (SEC) Procollagen Procollagen receptor MannoseMannose receptors N-acetyl Glucosamine Scavenger receptorsImmunoglobulins Fc Receptor LPS CD14 Receptor Insulin Receptor mediatedtranscytosis Transferrin Receptor mediated transcytosis AlbuminsNon-specific Sugar-Albumin conjugates Mannose-6-phosphateMannose-6-phosphate receptor 3) Kupffer Cell (KC) Mannose Mannosereceptors Fucose Fucose receptors Albumins Non-specific Mannose-albuminconjugates

Other examples of ligands include dyes, intercalating agents (e.g.acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins(TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA),lipophilic molecules, e.g, cholesterol, cholic acid, adamantane aceticacid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,bomeol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine)and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., aspirin, vitamin E, folicacid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a cancercell, endothelial cell, or bone cell. Ligands may also include hormonesand hormone receptors. They can also include non-peptidic species, suchas lipids, lectins, carbohydrates, vitamins, cofactors, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucosamine multivalent mannose, multivalent fucose, oraptamers. The ligand can be, for example, a lipopolysaccharide, anactivator of p38 MAP kinase, or an activator of NF-κB.

The ligand can be a substance, e.g, a drug, which can increase theuptake of the iRNA agent into the cell, for example, by disrupting thecell's cytoskeleton, e.g., by disrupting the cell's microtubules,microfilaments, and/or intermediate filaments. The drug can be, forexample, taxon, vincristine, vinblastine, cytochalasin, nocodazole,japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, ormyoservin.

The ligand can increase the uptake of the iRNA agent into the cell byactivating an inflammatory response, for example. Exemplary ligands thatwould have such an effect include tumor necrosis factor alpha(TNFalpha), interleukin-1 beta, or gamma interferon.

In one aspect, the ligand is a lipid or lipid-based molecule. Such alipid or lipid-based molecule preferably binds a serum protein, e.g.,human serum albumin (HSA). An HSA binding ligand allows for distributionof the conjugate to a target tissue, e.g., a non-kidney target tissue ofthe body. For example, the target tissue can be the liver, includingparenchymal cells of the liver. Other molecules that can bind HSA canalso be used as ligands. For example, neproxin or aspirin can be used. Alipid or lipid-based ligand can (a) increase resistance to degradationof the conjugate, (b) increase targeting or transport into a target cellor cell membrane, and/or (c) can be used to adjust binding to a serumprotein, e.g., HSA.

A lipid based ligand can be used to modulate, e.g., control the bindingof the conjugate to a target tissue. For example, a lipid or lipid-basedligand that binds to HSA more strongly will be less likely to betargeted to the kidney and therefore less likely to be cleared from thebody. A lipid or lipid-based ligand that binds to HSA less strongly canbe used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably,it binds HSA with a sufficient affinity such that the conjugate will bepreferably distributed to a non-kidney tissue. However, it is preferredthat the affinity not be so strong that the HSA-ligand binding cannot bereversed.

In another preferred embodiment, the lipid based ligand binds HSA weaklyor not at all, such that the conjugate will be preferably distributed tothe kidney. Other moieties that target to kidney cells can also be usedin place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include are B vitamin, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bycancer cells. Also included are HAS, low density lipoprotein (LDL) andhigh-density lipoprotein (HDL).

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as tat or antennopedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The peptide or peptidomimetic moiety can be about 5-50 aminoacids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 aminoacids long (see Table 3, for example).

TABLE 3 Exemplary Cell Permeation Peptides. Cell SEQ Permeation IDPeptide Amino acid Sequence Reference NO: Penetratin RQIKIWFQNRRMKWKKDerossi et al., J. Biol. 13 Chem. 269: 10444, 1994 Tat fragmentGRKKRRQRRRPPQC Vives et al., J. Biol. 14 (48-60) Chem., 272: 16010, 1997Signal GALFLGWLGAAGSTMGAWSQP Chaloin et al., 15 Sequence- KKKRKVBiochem. Biophys. based peptide Res. Commun., 243: 601, 1998 PVECLLIILRRRIRKQAHAHSK Elmquist et al., Exp. 16 Cell Res., 269: 237, 2001Transportan GWTLNSAGYLLKINLKALAALA Pooga et al., FASEB J., 17 KKIL12: 67, 1998 Amphiphilic KLALKLALKALKAALKLA Oehlke et al., Mol. 18model peptide Ther., 2: 339, 2000 Arg₉ RRRRRRRRRMitchell et al., J. Pept. 19 Res., 56: 318, 2000 Bacterial cellKFFKFFKFFK 20 wall permeating LL-37 LLGDFFRKSKEKIGKEFKRIVQR 21IKDFLRNLVPRTES Cecropin P1 SWLSKTAKKLENSAKKRISEGIA 22 IAIQGGPRα-defensin ACYCRIPACIAGERRYGTCIYQG 23 RLWAFCC b-defensinDHYNCVSSGGQCLYSACPIFTKI 24 QGTCYRGKAKCCK Bactenecin RKCRIVVIRVCR 25PR-39 RRRPRPPYLPRPRPPPFFPPRLPP 26 RIPPGFPPRFPPRFPGKR-NH2 IndolicidinILPWKWPWWPWRR-NH2 27

A peptide or peptidomimetic can be, for example, a cell permeationpeptide, cationic peptide, amphipathic peptide, or hydrophobic peptide(e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety canbe a dendrimer peptide, constrained peptide or crosslinked peptide. Inanother alternative, the peptide moiety can include a hydrophobicmembrane translocation sequence (MTS). An exemplary hydrophobicMTS-containing peptide is RFGF having the amino acid sequenceAAVALLPAVLLALLAP (SEQ ID NO: 28). An RFGF analogue (e.g., amino acidsequence AALLPVLLAAP (SEQ ID NO: 29)) containing a hydrophobic MTS canalso be a targeting moiety. The peptide moiety can be a “delivery”peptide, which can carry large polar molecules including peptides,oligonucleotides, and protein across cell membranes. For example,sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 14)) andthe Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 13))have been found to be capable of functioning as delivery peptides. Apeptide or peptidomimetic can be encoded by a random sequence of DNA,such as a peptide identified from a phage-display library, orone-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature,354:82-84, 1991). Preferably the peptide or peptidomimetic tethered toan iRNA agent via an incorporated monomer unit is a cell targetingpeptide such as an arginine-glycine-aspartic acid (RGD)-peptide, or RGDmimic. A peptide moiety can range in length from about 5 amino acids toabout 40 amino acids. The peptide moieties can have a structuralmodification, such as to increase stability or direct conformationalproperties. Any of the structural modifications described below can beutilized.

An RGD peptide moiety can be used to target a tumor cell, such as anendothelial tumor cell or a breast cancer tumor cell (Zitzmann et al.,Cancer Res., 62:5139-43, 2002). An RGD peptide can facilitate targetingof an iRNA agent to tumors of a variety of other tissues, including thelung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy8:783-787, 2001). Preferably, the RGD peptide will facilitate targetingof an iRNA agent to the kidney. The RGD peptide can be linear or cyclic,and can be modified, e.g., glycosylated or methylated to facilitatetargeting to specific tissues. For example, a glycosylated RGD peptidecan deliver an iRNA agent to a tumor cell expressing α_(v)β₃ (Haubner etal., Jour. Nucl. Med., 42:326-336, 2001).

Peptides that target markers enriched in proliferating cells can beused. E.g., RGD containing peptides and peptidomimetics can targetcancer cells, in particular cells that exhibit an I_(v)θ₃ integrin.Thus, one could use RGD peptides, cyclic peptides containing RGD, RGDpeptides that include D-amino acids, as well as synthetic RGD mimics. Inaddition to RGD, one can use other moieties that target the I_(v)-θ₃integrin ligand. Generally, such ligands can be used to controlproliferating cells and angiogeneis. Preferred conjugates of this typelignads that targets PECAM-1, VEGF, or other cancer gene, e.g., a cancergene described herein.

A “cell permeation peptide” is capable of permeating a cell, e.g., amicrobial cell, such as a bacterial or fungal cell, or a mammalian cell,such as a human cell. A microbial cell-permeating peptide can be, forexample, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), adisulfide bond-containing peptide (e.g., α-defensin, β-defensin orbactenecin), or a peptide containing only one or two dominating aminoacids (e.g., PR-39 or indolicidin). A cell permeation peptide can alsoinclude a nuclear localization signal (NLS). For example, a cellpermeation peptide can be a bipartite amphipathic peptide, such as MPG,which is derived from the fusion peptide domain of HIV-1 gp41 and theNLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.31:2717-2724, 2003).

In one embodiment, a targeting peptide can be an amphipathic α-helicalpeptide. Exemplary amphipathic α-helical peptides include, but are notlimited to, cecropins, lycotoxins, paradaxins, buforin, CPF,bombinin-like peptide (BLP), cathelicidins, ceratotoxins, S. clavapeptides, hagfish intestinal antimicrobial peptides (HFIAPs),magainines, brevinins-2, dermaseptins, melittins, pleurocidin, H₂Apeptides, Xenopus peptides, esculentinis-1, and caerins. A number offactors will preferably be considered to maintain the integrity of helixstability. For example, a maximum number of helix stabilization residueswill be utilized (e.g., leu, ala, or lys), and a minimum number helixdestabilization residues will be utilized (e.g., proline, or cyclicmonomeric units. The capping residue will be considered (for example Glyis an exemplary N-capping residue and/or C-terminal amidation can beused to provide an extra H-bond to stabilize the helix. Formation ofsalt bridges between residues with opposite charges, separated by i±3,or i±4 positions can provide stability. For example, cationic residuessuch as lysine, arginine, homo-arginine, ornithine or histidine can formsalt bridges with the anionic residues glutamate or aspartate.

Peptide and peptidomimetic ligands include those having naturallyoccurring or modified peptides, e.g., D or L peptides; α, β, or γpeptides; N-methyl peptides; azapeptides; peptides having one or moreamide, i.e., peptide, linkages replaced with one or more urea, thiourea,carbamate, or sulfonyl urea linkages; or cyclic peptides.

The targeting ligand can be any ligand that is capable of targeting aspecific receptor. Examples are: folate, GalNAc, galactose, mannose,mannose-6P, clusters of sugars such as GalNAc cluster, mannose cluster,galactose cluster, or an apatamer. A cluster is a combination of two ormore sugar units. The targeting ligands also include integrin receptorligands, Chemokine receptor ligands, transferrin, biotin, serotoninreceptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL and HDLligands. The ligands can also be based on nucleic acid, e.g., anaptamer. The aptamer can be unmodified or have any combination ofmodifications disclosed herein.

Endosomal release agents include imidazoles, poly or oligoimidazoles,PEIs, peptides, fusogenic peptides, polycarboxylates, polyacations,masked oligo or poly cations or anions, acetals, polyacetals,ketals/polyketyals, orthoesters, polymers with masked or unmaskedcationic or anionic charges, dendrimers with masked or unmasked cationicor anionic charges.

PK modulator stands for pharmacokinetic modulator. PK modulator includelipophiles, bile acids, steroids, phospholipid analogues, peptides,protein binding agents, PEG, vitamins etc. Examplary PK modulatorinclude, but are not limited to, cholesterol, fatty acids, cholic acid,lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids,sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc.Oligonucleotides that comprise a number of phosphorothioate linkages arealso known to bind to serum protein, thus short oligonucleotides, e.g.oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases,comprising multiple of phosphorothioate linkages in the backbaone arealso amenable to the present invention as ligands (e.g. as PK modulatingligands).

In addition, aptamers that bind serum components (e.g. serum proteins)are also amenable to the present invention as PK modulating ligands.

Other ligands amenable to the invention are described in copendingapplications U.S. Ser. No. 10/916,185, filed Aug. 10, 2004; U.S. Ser.No. 10/946,873, filed Sep. 21, 2004; U.S. Ser. No. 10/833,934, filedAug. 3, 2007; U.S. Ser. No. 11/115,989 filed Apr. 27, 2005 and U.S. Ser.No. 11/944,227 filed Nov. 21, 2007, which are incorporated by referencein their entireties for all purposes.

When two or more ligands are present, the ligands can all have sameproperties, all have different properties or some ligands have the sameproperties while others have different properties. For example, a ligandcan have targeting properties, have endosomolytic activity or have PKmodulating properties. In a preferred embodiment, all the ligands havedifferent properties.

Ligands can be coupled to the oligonucleotides at various places, forexample, 3′-end, 5′-end, and/or at an internal position. In preferredembodiments, the ligand is attached to the oligonucleotides via anintervening tether, e.g. a carrier described herein.

The ligand or tethered ligand may be present on a monomer when saidmonomer is incorporated into the growing strand. In some embodiments,the ligand may be incorporated via coupling to a “precursor” monomerafter said “precursor” monomer has been incorporated into the growingstrand. For example, a monomer having, e.g., an amino-terminated tether(i.e., having no associated ligand), e.g., TAP-(CH₂)NH₂ may beincorporated into a growing oligonucleotide strand. In a subsequentoperation, i.e., after incorporation of the precursor monomer into thestrand, a ligand having an electrophilic group, e.g., apentafluorophenyl ester or aldehyde group, can subsequently be attachedto the precursor monomer by coupling the electrophilic group of theligand with the terminal nucleophilic group of the precursor monomer'stether.

In another example, a monomer having a chemical group suitable fortaking part in Click Chemistry reaction may be incorporated e.g., anazide or alkyne terminated tether/linker. In a subsequent operation,i.e., after incorporation of the precursor monomer into the strand, aligand having complementary chemical group, e.g. an alkyne or azide canbe attached to the precursor monomer by coupling the alkyne and theazide together.

For double-stranded oligonucleotides, ligands can be attached to one orboth strands. In some embodiments, a double-stranded iRNA agent containsa ligand conjugated to the sense strand. In other embodiments, adouble-stranded iRNA agent contains a ligand conjugated to the antisensestrand.

In some embodiments, ligand can be conjugated to nucleobases, sugarmoieties, or internucleosidic linkages of nucleic acid molecules.Conjugation to purine nucleobases or derivatives thereof can occur atany position including, endocyclic and exocyclic atoms. In someembodiments, the 2-, 6-, 7-, or 8-positions of a purine nucleobase areattached to a conjugate moiety. Conjugation to pyrimidine nucleobases orderivatives thereof can also occur at any position. In some embodiments,the 2-, 5-, and 6-positions of a pyrimidine nucleobase can besubstituted with a conjugate moiety. Conjugation to sugar moieties ofnucleosides can occur at any carbon atom. Example carbon atoms of asugar moiety that can be attached to a conjugate moiety include the 2′,3′, and 5′ carbon atoms. The 1′ position can also be attached to aconjugate moiety, such as in an abasic residue. Internucleosidiclinkages can also bear conjugate moieties. For phosphorus-containinglinkages (e.g., phosphodiester, phosphorothioate, phosphorodithiotate,phosphoroamidate, and the like), the conjugate moiety can be attacheddirectly to the phosphorus atom or to an O, N, or S atom bound to thephosphorus atom. For amine- or amide-containing internucleosidiclinkages (e.g., PNA), the conjugate moiety can be attached to thenitrogen atom of the amine or amide or to an adjacent carbon atom.

There are numerous methods for preparing conjugates of oligomericcompounds. Generally, an oligomeric compound is attached to a conjugatemoiety by contacting a reactive group (e.g., OH, SH, amine, carboxyl,aldehyde, and the like) on the oligomeric compound with a reactive groupon the conjugate moiety. In some embodiments, one reactive group iselectrophilic and the other is nucleophilic.

For example, an electrophilic group can be a carbonyl-containingfunctionality and a nucleophilic group can be an amine or thiol. Methodsfor conjugation of nucleic acids and related oligomeric compounds withand without linking groups are well described in the literature such as,for example, in Manoharan in Antisense Research and Applications, Crookeand LeBleu, eds., CRC Press, Boca Raton, Fla., 1993, Chapter 17, whichis incorporated herein by reference in its entirety.

Representative United States patents that teach the preparation ofoligonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904, 582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,149,782; 5,214,136;5,245,022; 5,254, 469; 5,258,506; 5,262,536; 5,272,250; 5,292,873;5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475;5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574, 142; 5,585,481;5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928; 5,672,662;5,688,941; 5,714,166; 6,153,737; 6,172,208; 6,300,319; 6,335,434;6,335,437; 6,395,437; 6,444,806; 6,486,308; 6,525,031; 6,528,631;6,559,279; each of which is herein incorporated by reference.

The monomers and methods described herein can be used in the preparationof modified RNA, e.g., an iRNA agent, which incorporates a RRMS, such asthose described herein and those described in copending co-owned U.S.application Ser. Nos. 10/916,185 filed Aug. 10, 2004, 10/946,873 filedSep. 21, 2004, 10/985,426 filed Nov. 9, 2004 and 11/833,934 filed Aug.3, 2007, all of which are hereby incorporated by reference.

Modified RNA molecules include e.g. those molecules containing achemically or stereochemically modified nucleoside (e.g., having one ormore backbone modifications, e.g., phosphorothioate or P-alkyl; havingone or more sugar modifications, e.g., 2′-OCH₃ or 2′-F; and/or havingone or more base modifications, e.g., 5-alkylamino or 5-allylamino) or anucleoside surrogate.

Coupling of 5′-hydroxyl groups with phosphoramidites forms phosphiteester intermediates, which in turn are oxidized e.g., with iodine, tothe phosphate diester. Alternatively, the phosphites may be treated withe.g., sulfur, selenium, amino, and boron reagents to form modifiedphosphate backbones. Linkages between the monomers described herein anda nucleoside or oligonucleotide chain can also be treated with iodine,sulfur, selenium, amino, and boron reagents to form unmodified andmodified phosphate backbones respectively. Similarly, the monomersdescribed herein may be coupled with nucleosides or oligonucleotidescontaining any of the modifications or nucleoside surrogates describedherein.

The synthesis and purification of oligonucleotide ligand conjugates canbe performed by established methods. See, for example, Trufert et al.,Tetrahedron, 52:3005, 1996; and Manoharan, “Oligonucleotide Conjugatesin Antisense Technology,” in Antisense Drug Technology, ed. S. T.Crooke, Marcel Dekker, Inc., 2001.

iRNA Agent Structure

The monomers described herein can be used to make oligonucleotides whichare useful as iRNA agents, e.g., RNA molecules, (double-stranded;single-stranded) that mediate RNAi, e.g., with respect to an endogenousgene of a subject or to a gene of a pathogen. In most cases the iRNAagent will incorporate momomers described herein together with naturallyoccurring nucleosides or nucleotides or with other modified nucleosidesor nucleotides. The modified monomers can be present at any position inthe iRNA agent, e.g., at the terminii or in the middle region of an iRNAagent or in a duplex region or in an unpaired region. In a preferredembodiment iRNA agent can have any architecture, e.g., architecturedescribed herein. e.g., it can be incorporated into an iRNA agent havingan overhang structure, a hairpin or other single strand structure or atwo-strand structure, as described herein.

An “RNA agent” as used herein, is an unmodified RNA, modified RNA, ornucleoside surrogate, all of which are defined herein (see, e.g., thesection below entitled RNA Agents). While numerous modified RNAs andnucleoside surrogates are described, preferred examples include thosewhich have greater resistance to nuclease degradation than do unmodifiedRNAs. Preferred examples include those which have a 2′ sugarmodification, a modification in a single strand overhang, preferably a3′ single strand overhang, or, particularly if single stranded, a 5′modification which includes one or more phosphate groups or one or moreanalogs of a phosphate group.

An “iRNA agent” as used herein, is an RNA agent which can, or which canbe cleaved into an RNA agent which can, down regulate the expression ofa target gene, preferably an endogenous or pathogen target RNA. Whilenot wishing to be bound by theory, an iRNA agent may act by one or moreof a number of mechanisms, including post-transcriptional cleavage of atarget mRNA sometimes referred to in the art as RNAi, orpre-transcriptional or pre-translational mechanisms. An iRNA agent caninclude a single strand or can include more than one strands, e.g., itcan be a double stranded iRNA agent. If the iRNA agent is a singlestrand it is particularly preferred that it include a 5′ modificationwhich includes one or more phosphate groups or one or more analogs of aphosphate group.

The RRMS-containing iRNA agent should include a region of sufficienthomology to the target gene, and be of sufficient length in terms ofnucleotides, such that the iRNA agent, or a fragment thereof, canmediate down regulation of the target gene. (For ease of exposition theterm nucleotide or ribonucleotide is sometimes used herein in referenceto one or more monomeric subunits of an RNA agent. It will be understoodherein that the usage of the term “ribonucleotide” or “nucleotide”,herein can, in the case of a modified RNA or nucleotide surrogate, alsorefer to a modified nucleotide, or surrogate replacement moiety at oneor more positions.) Thus, the iRNA agent is or includes a region whichis at least partially, and in some embodiments fully, complementary tothe target RNA. It is not necessary that there be perfectcomplementarity between the iRNA agent and the target, but thecorrespondence must be sufficient to enable the iRNA agent, or acleavage product thereof, to direct sequence specific silencing, e.g.,by RNAi cleavage of the target RNA, e.g., mRNA.

As discussed elsewhere herein, an iRNA agent will often be modified orinclude nucleoside surrogates in addition to the ribose replacementmodification subunit (RRMS). Single stranded regions of an iRNA agentwill often be modified or include nucleoside surrogates, e.g., theunpaired region or regions of a hairpin structure, e.g., a region whichlinks two complementary regions, can have modifications or nucleosidesurrogates. Modification to stabilize one or more 3′- or 5′-terminus ofan iRNA agent, e.g., against exonucleases, or to favor the antisensestrand of the iRNA agent to enter into RISC are also favored.Modifications can include C3 (or C6, C7, C12) amino linkers, thiollinkers, carboxyl linkers, non-nucleotidic spacers (C3, C6, C9, C12,abasic, triethylene glycol, hexaethylene glycol), special biotin orfluorescein reagents that come as phosphoramidites and that have anotherDMT-protected hydroxyl group, allowing multiple couplings during RNAsynthesis.

In addition to homology to target RNA and the ability to down regulate atarget gene, an iRNA agent will preferably have one or more of thefollowing properties:

-   -   (1) if single stranded it will preferably have a 5′ modification        which includes one or more phosphate groups or one or more        analogs of a phosphate group;    -   (2) it will, despite modifications, even to a very large number,        or all of the nucleosides, have an antisense strand that can        present bases (or modified bases) in the proper three        dimensional framework so as to be able to form correct base        pairing and form a duplex structure with a homologous target RNA        which is sufficient to allow down regulation of the target,        e.g., by cleavage of the target RNA;    -   (3) it will, despite modifications, even to a very large number,        or all of the nucleosides, still have “RNA-like” properties,        i.e., it will possess the overall structural, chemical and        physical properties of an RNA molecule, even though not        exclusively, or even partly, of ribonucleotide-based content.        For example, an iRNA agent can contain, e.g., a sense and/or an        antisense strand in which all of the nucleotide sugars contain        e.g., 2′ fluoro in place of 2′ hydroxyl. This        deoxyribonucleotide-containing agent can still be expected to        exhibit RNA-like properties;    -   (4) regardless of the nature of the modification, and even        though the RNA agent can contain deoxynucleotides or modified        deoxynucleotides, particularly in overhang or other single        strand regions, it is preferred that DNA molecules, or any        molecule in which more than 50, 60, or 70% of the nucleotides in        the molecule, or more than 50, 60, or 70% of the nucleotides in        a duplexed region are deoxyribonucleotides, or modified        deoxyribonucleotides which are deoxy at the 2′ position, are        excluded from the definition of RNA agent.

A “single strand iRNA agent” as used herein, is an iRNA agent which ismade up of a single molecule. It may include a duplexed region, formedby intra-strand pairing, e.g., it may be, or include, a hairpin orpan-handle structure. Single strand iRNA agents are preferably antisensewith regard to the target molecule. In preferred embodiments singlestrand iRNA agents are 5′ phosphorylated or include a phosphoryl analogat the 5′ prime terminus. 5′-phosphate modifications include those whichare compatible with RISC mediated gene silencing. Suitable modificationsinclude: 5′-monophosphate ((HO)₂(O)P—O-5′); 5′-diphosphate((HO)₂(O)P—O—P(HO)(O)—O-5′); 5′-triphosphate((HO)₂(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap (7-methylatedor non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-adenosine cap (Appp), and any modified or unmodified nucleotide capstructure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-monothiophosphate (phosphorothioate; (HO)₂(S)P—O-5′);5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′),5′-phosphorothiolate ((HO)₂(O)P—S-5′); any additional combination ofoxygen/sulfur replaced monophosphate, diphosphate and triphosphates(e.g. 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.),5′-phosphoramidates ((HO)₂(O)P—NH-5′, (HO)(NH₂)(O)P—O-5′),5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc.,e.g. RP(OH)(O)—O-5′-, (OH)₂(O)P-5′-CH₂—), 5′-alkyletherphosphonates(R=alkylether=methoxymethyl (MeOCH₂—), ethoxymethyl, etc., e.g.RP(OH)(O)—O-5′-). (These modifications can also be used with theantisense strand of a double stranded iRNA.)

It may be desirable to modify one or both of the antisense and sensestrands of a double strand iRNA agent. In some cases they will have thesame modification or the same class of modification but in other casesthe sense and antisense strand will have different modifications, e.g.,in some cases it is desirable to modify only the sense strand. It may bedesirable to modify only the sense strand, e.g., to inactivate it, e.g.,the sense strand can be modified in order to inactivate the sense strandand prevent formation of an active sRNA/protein or RISC. This can beaccomplished by a modification which prevents 5′-phosphorylation of thesense strand, e.g., by modification with a 5′-O-methyl ribonucleotide(see Nykänen et al., (2001) ATP requirements and small interfering RNAstructure in the RNA interference pathway. Cell 107, 309-321.) Othermodifications which prevent phosphorylation can also be used, e.g.,simply substituting the 5′-OH by H rather than O-Me. Alternatively, alarge bulky group may be added to the 5′-phosphate turning it into aphosphodiester linkage, though this may be less desirable asphosphodiesterases can cleave such a linkage and release a functionalsRNA 5′-end. Antisense strand modifications include 5′ phosphorylationas well as any of the other 5′ modifications discussed herein,particularly the 5′ modifications discussed above in the section onsingle stranded iRNA molecules.

In some cases the sense and the antisense strands will include differentmodifications. Multiple different modifications can be included on thesense and antisense strands. The modifications on each strand may differfrom each other, and may also differ from the various modifications onthe other strand. For example, the sense strand may have a modification,e.g., a modification described herein, and the antisense strand may havea different modification, e.g., a different modification describedherein. In other cases, one strand, such as the sense strand may havetwo different modifications, and the antisense strand may include amodification that differs from the at least two modifications on thesense strand.

It is preferred that the sense and antisense strands be chosen such thatthe double stranded iRNA agent includes a single strand or unpairedregion at one or both ends of the molecule. Thus, a double-stranded iRNAagent contains sense and antisense strands, preferable paired to containan overhang, e.g., one or two 5′ or 3′ overhangs but preferably a 3′overhang of 2-3 nucleotides. Most embodiments will have a 3′ overhang.Preferred sRNA agents will have single-stranded overhangs, preferably 3′overhangs, of 1 or preferably 2 or 3 nucleotides in length at each end.The overhangs can be the result of one strand being longer than theother, or the result of two strands of the same length being staggered.5′ ends are preferably phosphorylated.

Preferred lengths for the duplexed region is between 15 and 30, mostpreferably 18, 19, 20, 21, 22, and 23 nucleotides in length, e.g., inthe sRNA agent range discussed above. sRNA agents can resemble in lengthand structure the natural Dicer processed products from long dsRNAs.Embodiments in which the two strands of the sRNA agent are linked, e.g.,covalently linked are also included. Hairpin, or other single strandstructures which provide the required double stranded region, andpreferably a 3′ overhang are also within the invention.

The monomers and methods described herein can also be used in thepreparation of oligonucleotides other than iRNA agents. In the contextof this invention, the term “oligonucleotide” refers to a polymer oroligomer of nucleotide or nucleoside monomers consisting of naturallyoccurring bases, sugars and intersugar (backbone) linkages. The term“oligonucleotide” also includes polymers or oligomers comprisingnon-naturally occurring monomers, or portions thereof, which functionsimilarly. Such modified or substituted oligonucleotides are oftenpreferred over native forms because of properties such as, for example,enhanced cellular uptake and increased stability in the presence ofnucleases.

The oligonucleotides used herein can be single-stranded DNA or RNA, ordouble-stranded DNA or RNA, or DNA-RNA hybrids. Examples ofdouble-stranded DNA include structural genes, genes including controland termination regions, and self-replicating systems such as viral orplasmid DNA. Examples of double-stranded RNA include siRNA, iRNA agentsand other RNA interference reagents. Single-stranded nucleic acidsinclude, e.g., antisense oligonucleotides, ribozymes, microRNA, andtriplex-forming oligonucleotides. The oligonucleotides of this inventionmay include one or more of the oligonucleotide modifications describedherein.

Nucleic acids of the present invention may be of various lengths,generally dependent upon the particular form of nucleic acid. Forexample, in particular embodiments, plasmids or genes may be from about1,000 to 100,000 nucleotide residues in length. In particularembodiments, oligonucleotides may range from about 10 to 100 nucleotidesin length. In various related embodiments, oligonucleotides,single-stranded, double-stranded, and triple-stranded, may range inlength from about 10 to about 50 nucleotides, from about 20 o about 50nucleotides, from about 15 to about 30 nucleotides, from about 20 toabout 30 nucleotides in length.

In particular embodiments, an oligonucleotide (or a strand thereof) ofthe present invention specifically hybridizes to or is complementary toa target polynucleotide. “Specifically hybridizable” and “complementary”are terms which are used to indicate a sufficient degree ofcomplementarity such that stable and specific binding occurs between theDNA or RNA target and the oligonucleotide. It is understood that anoligonucleotide need not be 100% complementary to its target nucleicacid sequence to be specifically hybridizable. An oligonucleotide isspecifically hybridizable when binding of the oligonucleotide to thetarget interferes with the normal function of the target molecule tocause a loss of utility or expression therefrom, and there is asufficient degree of complementarity to avoid non-specific binding ofthe oligonucleotide to non-target sequences under conditions in whichspecific binding is desired, i.e., under physiological conditions in thecase of in vivo assays or therapeutic treatment, or, in the case of invitro assays, under conditions in which the assays are conducted. Thus,in other embodiments, this oligonucleotide includes 1, 2, or 3 basesubstitutions, e.g. mismatches, as compared to the region of a gene ormRNA sequence that it is targeting or to which it specificallyhybridizes. Some exemplary oligonucleotides include antisenseoligonucleotide, ribozymes, apatamers, microRNAs and antagomirs.

As nucleic acids are polymers of subunits or monomers, many of themodifications described below occur at a position which is repeatedwithin a nucleic acid, e.g., a modification of a base, or a phosphatemoiety, or the a non-linking O of a phosphate moiety. In some cases themodification will occur at all of the subject positions in the nucleicacid but in many, and infact in most cases it will not. By way ofexample, a modification may only occur at a 3′ or 5′ terminal position,may only occur in a terminal regions, e.g. at a position on a terminalnucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand. Amodification may occur in a double strand region, a single strandregion, or in both. A modification may occur only in the double strandregion of an RNA or may only occur in a single strand region of an RNA.E.g., a phosphorothioate modification at a non-linking O position mayonly occur at one or both termini, may only occur in a terminal regions,e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5,or 10 nucleotides of a strand, or may occur in double strand and singlestrand regions, particularly at termini. The 5′ end or ends can bephosphorylated.

In some embodiments it is particularly preferred, e.g., to enhancestability, to include particular bases in overhangs, or to includemodified nucleotides or nucleotide surrogates, in single strandoverhangs, e.g., in a 5′ or 3′ overhang, or in both. E.g., it can bedesirable to include purine nucleotides in overhangs. In someembodiments all or some of the bases in a 3′ or 5′ overhang will bemodified, e.g., with a modification described herein. Modifications caninclude, e.g., the use of modifications at the 2′ OH group of the ribosesugar, e.g., the use of deoxyribonucleotides, e.g., deoxythymidine,instead of ribonucleotides, and modifications in the phosphate group,e.g., phosphothioate modifications. Overhangs need not be homologouswith the target sequence.

Unmodified oligoribonucleotides may be less than optimal in someapplications, e.g., unmodified oligoribonucleotides can be prone todegradation by e.g., cellular nucleases. Nucleases can hydrolyze nucleicacid phosphodiester bonds. However, chemical modifications to one ormore of the RNA components can confer improved properties, and, e.g.,can render oligoribonucleotides more stable to nucleases. Unmodifiedoligoribonucleotides may also be less than optimal in terms of offeringtethering points for attaching ligands or other moieties to an iRNAagent.

Modified nucleic acids and nucleotide surrogates can include one or moreof:

-   -   (i) alteration of the backbone, e.g., replacement, of one or        both of the non-linking phosphate oxygens and/or of one or more        of the linking phosphate oxygens in the backbone. For simplicity        of terminology, except where otherwise noted, one of the three        non-linking oxygens at the 5′ end of a nucleic acid and at the        3′ end of a nucleic acid, are within the term “linking phosphate        oxygens” as used herein.);    -   (ii) alteration, e.g., replacement, of a constituent of the        ribose sugar, e.g., of the 2′ hydroxyl on the ribose sugar, or        wholesale replacement of the ribose sugar with a structure other        than ribose, e.g., as described herein;    -   (iii) wholesale replacement of the phosphate diester with        “dephospho” linkers;    -   (iv) modification or replacement of a naturally occurring base;    -   (v) replacement or modification of the ribose-phosphate backbone        (bracket II);    -   (vi) modification of the 3′ end or 5′ end of the RNA, e.g.,        removal, modification or replacement of a terminal phosphate        group or conjugation of a moiety, e.g. a fluorescently labeled        moiety, to either the 3′ or 5′ end of RNA.

The terms replacement, modification, alteration, and the like, as usedin this context, do not imply any process limitation, e.g., modificationdoes not mean that one must start with a reference or naturallyoccurring ribonucleic acid and modify it to produce a modifiedribonucleic acid bur rather modified simply indicates a difference froma naturally occurring molecule.

The Phosphate Group

The phosphate group is a negatively charged species. The charge isdistributed equally over the two non-linking oxygen atoms. However, thephosphate group can be modified by replacing one or both of the oxygenswith a different substituent. One result of this modification to RNAphosphate backbones can be increased resistance of theoligoribonucleotide to nucleolytic breakdown. Thus while not wishing tobe bound by theory, it can be desirable in some embodiments to introducealterations which result in either an uncharged linker or a chargedlinker with unsymmetrical charge distribution.

Examples of modified phosphate groups include phosphorothioate,phosphoroselenates, borano phosphates, borano phosphate esters, hydrogenphosphonates, phosphoroamidates, alkyl or aryl phosphonates andphosphotriesters. Phosphorodithioates have both non-linking oxygensreplaced by sulfur.

The phosphate group can be replaced by non-phosphorus containingconnectors While not wishing to be bound by theory, it is believed thatsince the charged phosphodiester group is the reaction center innucleolytic degradation, its replacement with neutral structural mimicsshould impart enhanced nuclease stability. Again, while not wishing tobe bound by theory, it can be desirable, in some embodiment, tointroduce alterations in which the charged phosphate group is replacedby a neutral moiety.

Examples of moieties which can replace the phosphate group includesiloxane, carbonate, carboxymethyl, carbamate, amide, thioether,ethylene oxide linker, sulfonate, sulfonamide, thioformacetal,formacetal, oxime, methyleneimino, methylenemethylimino,methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.Preferred replacements include the methylenecarbonylamino andmethylenemethylimino groups.

The Sugar Group

A modified RNA can include modification of all or some of the sugargroups of the ribonucleic acid. For example, the 2′ hydroxyl group (OH)can be modified or replaced with a number of different “oxy” or “deoxy”substituents. While not being bound by theory, enhanced stability isexpected since the hydroxyl can no longer be deprotonated to form a 2′alkoxide ion. The 2′ alkoxide can catalyze degradation by intramolecularnucleophilic attack on the linker phosphorus atom. Again, while notwishing to be bound by theory, it can be desirable to some embodimentsto introduce alterations in which alkoxide formation at the 2′ positionis not possible.

Examples of “oxy”-2′ hydroxyl group modifications include alkoxy oraryloxy (OR, e.g., R═H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl orsugar); polyethyleneglycols (PEG), O(CH₂CH₂O)_(n)CH₂CH₂OR; “locked”nucleic acids (LNA) in which the 2′ hydroxyl is connected, e.g., by amethylene bridge, to the 4′ carbon of the same ribose sugar; O-AMINE(AMINE=NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroaryl amino, or diheteroaryl amino, ethylene diamine,polyamino) and aminoalkoxy, O(CH₂)_(n)AMINE, (e.g., AMINE=NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino).It is noteworthy that oligonucleotides containing only the methoxyethylgroup (MOE), (OCH₂CH₂OCH₃, a PEG derivative), exhibit nucleasestabilities comparable to those modified with the robustphosphorothioate modification.

“Deoxy” modifications include hydrogen (i.e. deoxyribose sugars, whichare of particular relevance to the overhang portions of partially dsRNA); halo (e.g., fluoro); amino (e.g. NH₂; alkylamino, dialkylamino,heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroarylamino, or amino acid); NH(CH₂CH₂NH)_(n)CH₂CH₂-AMINE (AMINE=NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, or diheteroaryl amino), —NHC(O)R(R=alkyl, cycloalkyl,aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl;thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which maybe optionally substituted with e.g., an amino functionality. Preferredsubstitutents are 2′-methoxyethyl, 2′-OCH3, 2′-O-allyl, 2′-C— allyl, and2′-fluoro.

The sugar group can also contain one or more carbons that possess theopposite stereochemical configuration than that of the correspondingcarbon in ribose. Thus, a modified RNA can include nucleotidescontaining e.g., arabinose, as the sugar.

Modified RNAs can also include “abasic” sugars, which lack a nucleobaseat C-1′. These abasic sugars can also be further contain modificationsat one or more of the constituent sugar atoms.

To maximize nuclease resistance, the 2′ modifications can be used incombination with one or more phosphate linker modifications (e.g.,phosphorothioate). The so-called “chimeric” oligonucleotides are thosethat contain two or more different modifications.

The modification can also entail the wholesale replacement of a ribosestructure with another entity at one or more sites in the iRNA agent.

Replacement of Ribophosphate Backbone

Oligonucleotide-mimicking scaffolds can also be constructed wherein thephosphate linker and ribose sugar are replaced by nuclease resistantnucleoside or nucleotide surrogates. While not wishing to be bound bytheory, it is believed that the absence of a repetitively chargedbackbone diminishes binding to proteins that recognize polyanions (e.g.nucleases). Again, while not wishing to be bound by theory, it can bedesirable in some embodiment, to introduce alterations in which thebases are tethered by a neutral surrogate backbone.

Examples include the mophilino, cyclobutyl, pyrrolidine and peptidenucleic acid (PNA) nucleoside surrogates. A preferred surrogate is a PNAsurrogate.

Terminal Modifications

The 3′ and 5′ ends of an oligonucleotide can be modified. Suchmodifications can be at the 3′ end, 5′ end or both ends of the molecule.They can include modification or replacement of an entire terminalphosphate or of one or more of the atoms of the phosphate group. Forexample, the 3′ and 5′ ends of an oligonucleotide can be conjugated toother functional molecular entities such as labeling moieties, e.g.,fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) orprotecting groups (based e.g., on sulfur, silicon, boron or ester). Thefunctional molecular entities can be attached to the sugar through aphosphate group and/or a spacer. The terminal atom of the spacer canconnect to or replace the linking atom of the phosphate group or theC-3′ or C-5′ O, N, S or C group of the sugar. Alternatively, the spacercan connect to or replace the terminal atom of a nucleotide surrogate(e.g., PNAs). These spacers or linkers can include e.g., —(CH₂)_(n)—,—(CH₂)N—, —(CH₂)_(n)O—, —(CH₂)S—, O(CH₂CH₂O)CH₂CH₂OH (e.g., n=3 or 6),abasic sugars, amide, carboxy, amine, oxyamine, oxyimine, thioether,disulfide, thiourea, sulfonamide, or morpholino, or biotin andfluorescein reagents. When a spacer/phosphate-functional molecularentity-spacer/phosphate array is interposed between two strands of iRNAagents, this array can substitute for a hairpin RNA loop in ahairpin-type RNA agent. The 3′ end can be an —OH group. While notwishing to be bound by theory, it is believed that conjugation ofcertain moieties can improve transport, hybridization, and specificityproperties. Again, while not wishing to be bound by theory, it may bedesirable to introduce terminal alterations that improve nucleaseresistance. Other examples of terminal modifications include dyes,intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene,mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclicaromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificialendonucleases (e.g. EDTA), lipophilic carriers (e.g., cholesterol,cholic acid, adamantane acetic acid, 1-pyrene butyric acid,dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexylgroup, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecylgroup, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid,O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine)and peptideconjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents,phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂,polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes,haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin,vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole,bisimidazole, histamine, imidazole clusters, acridine-imidazoleconjugates, Eu3+ complexes of tetraazamacrocycles).

Terminal modifications can be added for a number of reasons, includingas discussed elsewhere herein to modulate activity or to modulateresistance to degradation. Terminal modifications useful for modulatingactivity include modification of the 5′ end with phosphate or phosphateanalogs. E.g., in preferred embodiments iRNA agents, especiallyantisense strands, are 5′ phosphorylated or include a phosphoryl analogat the 5′ prime terminus. 5′-phosphate modifications include those whichare compatible with RISC mediated gene silencing.

Terminal modifications can also be useful for monitoring distribution,and in such cases the preferred groups to be added include fluorophores,e.g., fluorscein or an Alexa dye, e.g., Alexa 488. Terminalmodifications can also be useful for enhancing uptake, usefulmodifications for this include cholesterol. Terminal modifications canalso be useful for cross-linking an RNA agent to another moiety;modifications useful for this include mitomycin C.

The Bases

Adenine, guanine, cytosine and uracil are the most common bases found inRNA. These bases can be modified or replaced to provide RNA's havingimproved properties. For example, nuclease resistantoligoribonucleotides can be prepared with these bases or with syntheticand natural nucleobases (e.g., inosine, thymine, xanthine, hypoxanthine,nubularine, isoguanisine, or tubercidine) and any one of the abovemodifications. Alternatively, substituted or modified analogs of any ofthe above bases, e.g., “unusual bases” and “universal bases” describedherein, can be employed. Examples include without limitation2-aminoadenine, 6-methyl and other alkyl derivatives of adenine andguanine, 2-propyl and other alkyl derivatives of adenine and guanine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil,5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo,amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines andguanines, 5-trifluoromethyl and other 5-substituted uracils andcytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidinesand N-2, N-6 and O-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine,dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil,7-alkylguanine, 5-alkyl cytosine, 7-deazaadenine, N6,N6-dimethyladenine,2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole,5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil,5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil,5-methylaminomethyl-2-thiouracil, 3-(3-amino-3-carboxypropyl)uracil,3-methylcytosine, 5-methylcytosine, N⁴-acetyl cytosine, 2-thiocytosine,N6-methyladenine, N6-isopentyladenine,2-methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylatedbases. Further purines and pyrimidines include those disclosed in U.S.Pat. No. 3,687,808, those disclosed in the Concise Encyclopedia OfPolymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed.John Wiley & Sons, 1990, and those disclosed by Englisch et al.,Angewandte Chemie, International Edition, 1991, 30, 613.

Generally, base changes are less preferred for promoting stability, butthey can be useful for other reasons, e.g., some, e.g.,2,6-diaminopurine and 2 amino purine, are fluorescent. Modified basescan reduce target specificity. This should be taken into considerationin the design of iRNA agents.

Exemplary Modifications and Placement within an iRNA Agent

Some modifications may preferably be included on an iRNA agent at aparticular location, e.g., on the sense strand or antisense strand, oron the 5′ or 3′ end of the sense or antisense strand of an iRNA agent. Apreferred location of a modification on an iRNA agent, may conferpreferred properties on the agent. For example, preferred locations ofparticular modifications may confer optimum gene silencing properties,or increased resistance to endonuclease or exonuclease activity. Amodification described herein and below may be the sole modification, orthe sole type of modification included on multiple ribonucleotides, or amodification can be combined with one or more other modificationsdescribed herein and below. For example, a modification on a sensestrand of a dsRNA agent can be different than a modification on theantisense strand of an iRNA agent. Similarly, two differentmodifications on the sense strand can differ from a modification on theantisense strand. Other additional unique modifications, withoutlimitation, can be incorporates into the sense and antisense strands.

An iRNA agent may include a backbone modification to any nucleotide onan iRNA strand. For example, an iRNA agent may include aphosphorothioate linkage or P-alkyl modification in the linkages betweenone or more nucleotides of an iRNA agent. The nucleotides can beterminal nucleotides, e.g., nucleotides at the last position of a senseor antisense strand, or internal nucleotides.

An iRNA agent can include a sugar modification, e.g., a 2′ or 3′ sugarmodification. Exemplary sugar modifications include, for example, a2′-O-methylated nucleotide, a 2′-deoxy nucleotide, (e.g., a2′-deoxyfluoro nucleotide), a 2′-O-methoxyethyl nucleotide, a 2′-O-NMA,a 2′-DMAEOE, a 2′-aminopropyl, 2′-hydroxy, or a 2′-ara-fluoro or alocked nucleic acid (LNA), extended nucleic acid (ENA), hexose nucleicacid (HNA), or cyclohexene nucleic acid (CeNA). A 2′ modification ispreferably 2′OMe, and more preferably, 2′-deoxyfluoro. When themodification is 2′OMe, the modification is preferably on the sensestrand. When the modification is a 2′ fluoro, and the modification maybe on the sense or antisense strand, or on both strands. A 2′-ara-fluoromodification will preferably be on the sense strand of the iRNA agent.An LNA modification will preferably be on the sense strand of the iRNAagent or on the

An iRNA agent may include a 3′ sugar modification, e.g., a 3′OMemodification. Preferably a 3′OMe modification is on the sense strand ofthe iRNA agent.

An iRNA agent may includes a 5′-methyl-pyrimidine (e.g., a5′-methyl-uridine modification or a 5′-methyl-cytodine) modification.

The modifications described herein can be combined onto a single iRNAagent. For example, an iRNA agent may have a phosphorothioate linkageand a 2′ sugar modification, e.g., a 2′OMe or 2′F modification. Inanother example, an iRNA agent may include at least one 5′ Me-pyrimidineand a 2′ sugar modification, e.g., a 2′F or 2′OMe modification.

An iRNA agent may include a nucleobase modification, such as a cationicmodification, such as a 3′-abasic cationic modification. The cationicmodification can be e.g., an alkylamino-dT (e.g., a C6 amino-dT), anallylamino conjugate, a pyrrolidine conjugate, a pthalamido, aporphyrin, or a hydroxyprolinol conjugate, on one or more of theterminal nucleotides of the iRNA agent. When an alkylamino-dT conjugateis attached to the terminal nucleotide of an iRNA agent, the conjugateis preferably attached to the 3′ end of the sense or antisense strand ofan iRNA agent. When a pyrrolidine linker is attached to the terminalnucleotide of an iRNA agent, the linker is preferably attached to the 3′or 5′ end of the sense strand, or the 3′ end of the antisense strand.When a pyrrolidine linker is attached to the terminal nucleotide of aniRNA agent, the linker is preferably on the 3′ or 5′ end of the sensestrand, and not on the 5′ end of the antisense strand.

One or more nucleotides of an iRNA agent may have a 2′-5′ linkage.Preferably, the 2′-5′ linkage is on the sense strand. When the 2′-5′linkage is on the terminal nucleotide of an iRNA agent, the 2′-5′linkage occurs on the 5′ end of the sense strand.

The iRNA agent may include an L-sugar, preferably on the sense strand,and not on the antisense strand.

The iRNA agent may include a methylphosphonate modification. When themethylphosphonate is on the terminal nucleotide of an iRNA agent, themethylphosphonate is at the 3′ end of the sense or antisense strands ofthe iRNA agent.

An iRNA agent may be modified by replacing one or more ribonucleotideswith deoxyribonucleotides. Preferably, adjacent deoxyribonucleotides arejoined by phosphorothioate linkages, and the iRNA agent does not includemore than four consecutive deoxyribonucleotides on the sense or theantisense strands.

An iRNA agent may include a difluorotoluoyl (DFT) modification, e.g.,2,4-difluorotoluoyl uracil, or a guanidine to inosine substitution.

The iRNA agent may include at least one 5′-uridine-adenine-3′ (5′-UA-3′)dinucleotide wherein the uridine is a 2′-modified nucleotide, or aterminal 5′-uridine-guanine-3′ (5′-UG-3′) dinucleotide, wherein the5′-uridine is a 2′-modified nucleotide, or a terminal5′-cytidine-adenine-3′ (5′-CA-3′) dinucleotide, wherein the 5′-cytidineis a 2′-modified nucleotide, or a terminal 5′-uridine-uridine-3′(5′-UU-3′) dinucleotide, wherein the 5′-uridine is a 2′-modifiednucleotide, or a terminal 5′-cytidine-cytidine-3′ (5′-CC-3′)dinucleotide, wherein the 5′-cytidine is a 2′-modified nucleotide, or aterminal 5′-cytidine-uridine-3′ (5′-CU-3′) dinucleotide, wherein the5′-cytidine is a 2′-modified nucleotide, or a terminal5′-uridine-cytidine-3′ (5′-UC-3′) dinucleotide, wherein the 5′-uridineis a 2′-modified nucleotide. The chemically modified nucleotide in theiRNA agent may be a 2′-O-methylated nucleotide. In some embodiments, themodified nucleotide can be a 2′-deoxy nucleotide, a 2′-deoxyfluoronucleotide, a 2′-O-methoxyethyl nucleotide, a 2′-O-NMA, a 2′-DMAEOE, a2′-aminopropyl, 2′-hydroxy, or a 2′-ara-fluoro, or a locked nucleic acid(LNA), extended nucleic acid (ENA), hexose nucleic acid (HNA), orcyclohexene nucleic acid (CeNA). The iRNA agents including thesemodifications are particularly stabilized against exonuclease activity,when the modified dinucleotide occurs on a terminal end of the sense orantisense strand of an iRNA agent, and are otherwise particularlystabilized against endonuclease activity.

An iRNA agent may have a single overhang, e.g., one end of the iRNAagent has a 3′ or 5′ overhang and the other end of the iRNA agent is ablunt end, or the iRNA agent may have a double overhang, e.g., both endsof the iRNA agent have a 3′ or 5′ overhang, such as a dinucleotideoverhang. In another alternative, both ends of the iRNA agent may haveblunt ends.

The iRNA agent may further include a sense RNA strand and an antisenseRNA strand, wherein the antisense RNA strand is 25 or fewer nucleotidesin length, and includes an antisense nucleotide sequence having 18-25nucleotides in length. The iRNA agent may further include a nucleotideoverhang having 1 to 4 unpaired nucleotides, which may be at the 3′-endof the antisense RNA strand, and the nucleotide overhang may have thenucleotide sequence 5′-GC-3′ or 5′-CGC-3′. The unpaired nucleotides mayhave at least one phosphorothioate dinucleotide linkage, and at leastone of the unpaired nucleotides may be chemically modified in the2′-position. The doublestrand region of the iRNA agent may includephosphorothioate dinucleotide linkages on one or both of the sense andantisense strands. The antisense RNA strand and the sense RNA strand maybe connected with a linker, e.g., a chemical linker such as hexaethyleneglycol linker, a poly-(oxyphosphinico-oxy-1,3-propandiol) linker, anallyl linker, or a polyethylene glycol linker.

REFERENCES General References

The oligoribonucleotides and oligoribonucleosides used in accordancewith this invention may be with solid phase synthesis, see for example“Oligonucleotide synthesis, a practical approach”, Ed. M. J. Gait, IRLPress, 1984; “Oligonucleotides and Analogues, A Practical Approach”, Ed.F. Eckstein, IRL Press, 1991 (especially Chapter 1, Modern machine-aidedmethods of oligodeoxyribonucleotide synthesis, Chapter 2,Oligoribonucleotide synthesis, Chapter3,2′-O-Methyloligoribonucleotide-s: synthesis and applications, Chapter4, Phosphorothioate oligonucleotides, Chapter 5, Synthesis ofoligonucleotide phosphorodithioates, Chapter 6, Synthesis ofoligo-2′-deoxyribonucleoside methylphosphonates, and. Chapter 7,Oligodeoxynucleotides containing modified bases. Other particularlyuseful synthetic procedures, reagents, blocking groups and reactionconditions are described in Martin, P., Helv. Chim. Acta, 1995, 78,486-504; Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1992, 48,2223-2311 and Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1993, 49,6123-6194, or references referred to therein.

Modification described in WO 00/44895, WO01/75164, or WOO02/44321 can beused herein.

The disclosure of all publications, patents, and published patentapplications listed herein are hereby incorporated by reference.

Phosphate Group References

The preparation of phosphinate oligoribonucleotides is described in U.S.Pat. No. 5,508,270. The preparation of alkyl phosphonateoligoribonucleotides is described in U.S. Pat. No. 4,469,863. Thepreparation of phosphoramidite oligoribonucleotides is described in U.S.Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878. The preparation ofphosphotriester oligoribonucleotides is described in U.S. Pat. No.5,023,243. The preparation of borano phosphate oligoribonucleotide isdescribed in U.S. Pat. Nos. 5,130,302 and 5,177,198. The preparation of3′-Deoxy-3′-amino phosphoramidate oligoribonucleotides is described inU.S. Pat. No. 5,476,925. 3′-Deoxy-3′-methylenephosphonateoligoribonucleotides is described in An, H, et al. J. Org. Chem. 2001,66, 2789-2801. Preparation of sulfur bridged nucleotides is described inSproat et al. Nucleosides Nucleotides 1988, 7,651 and Crosstick et al.Tetrahedron Lett. 1989, 30, 4693.

Sugar Group References

Modifications to the 2′ modifications can be found in Verma, S. et al.Annu. Rev. Biochem. 1998, 67, 99-134 and all references therein.Specific modifications to the ribose can be found in the followingreferences: 2′-fluoro (Kawasaki et. al., J. Med. Chem., 1993, 36,831-841), 2′-MOE (Martin, P. Helv. Chim. Acta 1996, 79, 1930-1938),“LNA” (Wengel, J. Acc. Chem. Res. 1999, 32, 301-310).

Replacement of the Phosphate Group References

Methylenemethylimino linked oligoribonucleosides, also identified hereinas MMI linked oligoribonucleosides, methylenedimethylhydrazo linkedoligoribonucleosides, also identified herein as MDH linkedoligoribonucleosides, and methylenecarbonylamino linkedoligonucleosides, also identified herein as amide-3 linkedoligoribonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified herein as amide-4 linkedoligoribonucleosides as well as mixed backbone compounds having, as forinstance, alternating MMI and PO or PS linkages can be prepared as isdescribed in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677 and inpublished PCT applications PCT/US92/04294 and PCT/US92/04305 (publishedas WO 92/20822 WO and 92/20823, respectively). Formacetal andthioformacetal linked oligoribonucleosides can be prepared as isdescribed in U.S. Pat. Nos. 5,264,562 and 5,264,564. Ethylene oxidelinked oligoribonucleosides can be prepared as is described in U.S. Pat.No. 5,223,618. Siloxane replacements are described in Cormier, J. F. etal. Nucleic Acids Res. 1988, 16, 4583. Carbonate replacements aredescribed in Tittensor, J. R. J. Chem. Soc. C 1971, 1933. Carboxymethylreplacements are described in Edge, M. D. et al. J. Chem. Soc. PerkinTrans. 11972, 1991. Carbamate replacements are described in Stirchak, E.P. Nucleic Acids Res. 1989, 17, 6129.

Replacement of the Phosphate-Ribose Backbone References

Cyclobutyl sugar surrogate compounds can be prepared as is described inU.S. Pat. No. 5,359,044. Pyrrolidine sugar surrogate can be prepared asis described in U.S. Pat. No. 5,519,134. Morpholino sugar surrogates canbe prepared as is described in U.S. Pat. Nos. 5,142,047 and 5,235,033,and other related patent disclosures. Peptide Nucleic Acids (PNAs) areknown per se and can be prepared in accordance with any of the variousprocedures referred to in Peptide Nucleic Acids (PNA): Synthesis,Properties and Potential Applications, Bioorganic & Medicinal Chemistry,1996, 4, 5-23. They may also be prepared in accordance with U.S. Pat.No. 5,539,083.

Terminal Modification References

Terminal modifications are described in Manoharan, M. et al. Antisenseand Nucleic Acid Drug Development 12, 103-128 (2002) and referencestherein.

Bases References

N-2 substituted purine nucleoside amidites can be prepared as isdescribed in U.S. Pat. No. 5,459,255. 3-Deaza purine nucleoside amiditescan be prepared as is described in U.S. Pat. No. 5,457,191.5,6-Substituted pyrimidine nucleoside amidites can be prepared as isdescribed in U.S. Pat. No. 5,614,617. 5-Propynyl pyrimidine nucleosideamidites can be prepared as is described in U.S. Pat. No. 5,484,908.Additional references can be disclosed in the above section on basemodifications.

Nuclease Resistant Monomers

An iRNA agent can include monomers which have been modified so as toinhibit degradation, e.g., by nucleases, e.g., endonucleases orexonucleases, found in the body of a subject. These monomers arereferred to herein as NRMs, or nuclease resistance promoting monomers ormodifications. In many cases these modifications will modulate otherproperties of the iRNA agent as well, e.g., the ability to interact witha protein, e.g., a transport protein, e.g., serum albumin, or a memberof the RISC(RNA-induced Silencing Complex), or the ability of the firstand second sequences to form a duplex with one another or to form aduplex with another sequence, e.g., a target molecule.

The monomers and methods described herein can be used to prepare an RNA,e.g., an iRNA agent, that incorporates a nuclease resistant monomer(NRM), such as those described herein and those described in copending,co-owned U.S. Provisional application Ser. No. 10/553,659 filed on Apr.14, 2006, and International Application No. PCT/US04/07070, both ofwhich are hereby incorporated by reference.

Linkers

The term “linker” means an organic moiety that connects two parts of acompound. Linkers typically comprise a direct bond or an atom such asoxygen or sulfur, a unit such as NR¹, C(O), C(O)NH, SO, SO₂, SO₂NH or achain of atoms, such as substituted or unsubstituted alkyl, substitutedor unsubstituted alkenyl, substituted or unsubstituted alkynyl,arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl,heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl,heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl,cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl,alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl,alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl,alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl,alkenylheteroarylalkyl, alkenylheteroarylalkenyl,alkenylheteroarylalkynyl, alkynylheteroarylalkyl,alkynylheteroarylalkenyl, alkynylheteroarylalkynyl,alkylheterocyclylalkyl, alkylheterocyclylalkenyl,alkylhererocyclylalkynyl, alkenylheterocyclylalkyl,alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl,alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl,alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl,alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, where one or moremethylenes can be interrupted or terminated by O, S, S(O), SO₂, N(R¹)₂,C(O), cleavable linking group, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedheterocyclic; where R¹ is hydrogen, acyl, aliphatic or substitutedaliphatic. It is further understood that the term “linker” alsoencompasses tethers. In one embodiment, the linker is—[(P-Q″—R)_(q)—X—(P′-Q′″-R′)_(q′)]_(q″)-T-, wherein:

P, R, T, P′, R′ and T are each independently for each occurrence absent,CO, NH, O, S, OC(O), NHC(O), CH₂, CH₂NH, CH₂OO; NHCH(R^(a))C(O),—C(O)—CH(R^(a))—NH—, CH═N—O,

or heterocyclyl;

Q″ and Q′″ are each independently for each occurrence absent,—(CH₂)_(n)—, —C(R¹)(R²)(CH₂)_(n)—, —(CH₂)_(n)C(R¹)(R²)—,—(CH₂CH₂O)_(m)CH₂CH₂—, or —(CH₂CH₂O)_(m)CH₂CH₂NH—;

X is absent or a cleavable linking group;

R^(a) is H or an amino acid side chain;

R¹ and R² are each independently for each occurrence H, CH₃, OH, SH orN(R^(N))₂;

R^(N) is independently for each occurrence H, methyl, ethyl, propyl,isopropyl, butyl or benzyl;

q, q′ and q″ are each independently for each occurrence 0-20 and whereinthe repeating unit can be the same or different;

n is independently for each occurrence 1-20; and

m is independently for each occurrence 0-50.

In one embodiment, the linker comprises at least one cleavable linkinggroup.

In certain embodiments, the linker is a branched linker. The branchpointof the branched linker may be at least trivalent, but may be atetravalent, pentavalent or hexavalent atom, or a group presenting suchmultiple valencies. In certain embodiments, the branchpoint is, —N,—N(O)—C, —O—C, —S—C, —SS—C, —C(O)N(O)—C, —OC(O)N(O)—C, —N(O)C(O)—C, or—N(O)C(O)O—C; wherein Q is independently for each occurrence H oroptionally substituted alkyl. In other embodiment, the branchpoint isglycerol or glycerol derivative.

In certain embodiments, the linker is a branched linker. The branchpointof the branched linker may be at least trivalent, but may be atetravalent, pentavalent or hexavalent atom, or a group presenting suchmultiple valencies. In certain embodiments, the branchpoint is, —N,—N(O)—C, —O—C, —S—C, —SS—C, —C(O)N(O)—C, —OC(O)N(O)—C, —N(O)C(O)—C, or—N(O)C(O)O—C; wherein Q is independently for each occurrence H oroptionally substituted alkyl. In other embodiment, the branchpoint isglycerol or glycerol derivative.

Cleavable Linking Groups

A cleavable linking group is one which is sufficiently stable outsidethe cell, but which upon entry into a target cell is cleaved to releasethe two parts the linker is holding together. In a preferred embodiment,the cleavable linking group is cleaved at least 10 times or more,preferably at least 100 times faster in the target cell or under a firstreference condition (which can, e.g., be selected to mimic or representintracellular conditions) than in the blood of a subject, or under asecond reference condition (which can, e.g., be selected to mimic orrepresent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH,redox potential or the presence of degradative molecules. Generally,cleavage agents are more prevalent or found at higher levels oractivities inside cells than in serum or blood. Examples of suchdegradative agents include: redox agents which are selected forparticular substrates or which have no substrate specificity, including,e.g., oxidative or reductive enzymes or reductive agents such asmercaptans, present in cells, that can degrade a redox cleavable linkinggroup by reduction; esterases; endosomes or agents that can create anacidic environment, e.g., those that result in a pH of five or lower;enzymes that can hydrolyze or degrade an acid cleavable linking group byacting as a general acid, peptidases (which can be substrate specific),and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptibleto pH. The pH of human serum is 7.4, while the average intracellular pHis slightly lower, ranging from about 7.1-7.3. Endosomes have a moreacidic pH, in the range of 5.5-6.0, and lysosomes have an even moreacidic pH at around 5.0. Some linkers will have a cleavable linkinggroup that is cleaved at a preferred pH, thereby releasing the cationiclipid from the ligand inside the cell, or into the desired compartmentof the cell.

A linker can include a cleavable linking group that is cleavable by aparticular enzyme. The type of cleavable linking group incorporated intoa linker can depend on the cell to be targeted. For example, livertargeting ligands can be linked to the cationic lipids through a linkerthat includes an ester group. Liver cells are rich in esterases, andtherefore the linker will be cleaved more efficiently in liver cellsthan in cell types that are not esterase-rich. Other cell-types rich inesterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell typesrich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group canbe evaluated by testing the ability of a degradative agent (orcondition) to cleave the candidate linking group. It will also bedesirable to also test the candidate cleavable linking group for theability to resist cleavage in the blood or when in contact with othernon-target tissue. Thus one can determine the relative susceptibility tocleavage between a first and a second condition, where the first isselected to be indicative of cleavage in a target cell and the second isselected to be indicative of cleavage in other tissues or biologicalfluids, e.g., blood or serum. The evaluations can be carried out in cellfree systems, in cells, in cell culture, in organ or tissue culture, orin whole animals. It may be useful to make initial evaluations incell-free or culture conditions and to confirm by further evaluations inwhole animals. In preferred embodiments, useful candidate compounds arecleaved at least 2, 4, 10 or 100 times faster in the cell (or under invitro conditions selected to mimic intracellular conditions) as comparedto blood or serum (or under in vitro conditions selected to mimicextracellular conditions).

Redox Cleavable Linking Groups

One class of cleavable linking groups are redox cleavable linking groupsthat are cleaved upon reduction or oxidation. An example of reductivelycleavable linking group is a disulphide linking group (—S—S—). Todetermine if a candidate cleavable linking group is a suitable“reductively cleavable linking group,” or for example is suitable foruse with a particular iRNA moiety and particular targeting agent one canlook to methods described herein. For example, a candidate can beevaluated by incubation with dithiothreitol (DTT), or other reducingagent using reagents know in the art, which mimic the rate of cleavagewhich would be observed in a cell, e.g., a target cell. The candidatescan also be evaluated under conditions which are selected to mimic bloodor serum conditions. In a preferred embodiment, candidate compounds arecleaved by at most 10% in the blood. In preferred embodiments, usefulcandidate compounds are degraded at least 2, 4, 10 or 100 times fasterin the cell (or under in vitro conditions selected to mimicintracellular conditions) as compared to blood (or under in vitroconditions selected to mimic extracellular conditions). The rate ofcleavage of candidate compounds can be determined using standard enzymekinetics assays under conditions chosen to mimic intracellular media andcompared to conditions chosen to mimic extracellular media.

Phosphate-Based Cleavable Linking Groups

Phosphate-based cleavable linking groups are cleaved by agents thatdegrade or hydrolyze the phosphate group. An example of an agent thatcleaves phosphate groups in cells are enzymes such as phosphatases incells. Examples of phosphate-based linking groups are —O—P(O)(ORk)-O—,—O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—,—S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O—P(O)(Rk)—O—,—O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—,—O—P(S)(Rk)-S—. Preferred embodiments are —O—P(O)(OH)—O—,—O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—,—S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—,—O—P(S)(H)—O—, —S—P(O)(H)—O—, —S—P(S)(H)—O—, —S—P(O)(H)—S—,—O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—. Thesecandidates can be evaluated using methods analogous to those describedabove.

Acid Cleavable Linking Groups

Acid cleavable linking groups are linking groups that are cleaved underacidic conditions. In preferred embodiments acid cleavable linkinggroups are cleaved in an acidic environment with a pH of about 6.5 orlower (e.g., about 6.0, 5.5, 5.0, or lower), or by agents such asenzymes that can act as a general acid. In a cell, specific low pHorganelles, such as endosomes and lysosomes can provide a cleavingenvironment for acid cleavable linking groups. Examples of acidcleavable linking groups include but are not limited to hydrazones,esters, and esters of amino acids. Acid cleavable groups can have thegeneral formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is whenthe carbon attached to the oxygen of the ester (the alkoxy group) is anaryl group, substituted alkyl group, or tertiary alkyl group such asdimethyl pentyl or t-butyl. These candidates can be evaluated usingmethods analogous to those described above.

Ester-Based Linking Groups

Ester-based cleavable linking groups are cleaved by enzymes such asesterases and amidases in cells. Examples of ester-based cleavablelinking groups include but are not limited to esters of alkylene,alkenylene and alkynylene groups. Ester cleavable linking groups havethe general formula —C(O)O—, or —OC(O)—. These candidates can beevaluated using methods analogous to those described above.

Peptide-Based Cleaving Groups

Peptide-based cleavable linking groups are cleaved by enzymes such aspeptidases and proteases in cells. Peptide-based cleavable linkinggroups are peptide bonds formed between amino acids to yieldoligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides.Peptide-based cleavable groups do not include the amide group(—C(O)NH—). The amide group can be formed between any alkylene,alkenylene or alkynelene. A peptide bond is a special type of amide bondformed between amino acids to yield peptides and proteins. The peptidebased cleavage group is generally limited to the peptide bond (i.e., theamide bond) formed between amino acids yielding peptides and proteinsand does not include the entire amide functional group. Peptide-basedcleavable linking groups have the general formula—NHCHR^(A)C(O)NHCHR^(B)C(O)—, where R^(A) and R^(B) are the R groups ofthe two adjacent amino acids. These candidates can be evaluated usingmethods analogous to those described above.

Synthesis

As can be appreciated by the skilled artisan, methods of synthesizingthe compounds of the formulae herein will be evident to those ofordinary skill in the art. The synthesized compounds can be separatedfrom a reaction mixture and further purified by a method such as columnchromatography, high pressure liquid chromatography, orrecrystallization. Additionally, the various synthetic steps may beperformed in an alternate sequence or order to give the desiredcompounds. Synthetic chemistry transformations and protecting groupmethodologies (protection and deprotection) useful in synthesizing thecompounds described herein are known in the art and include, forexample, those such as described in R. Larock, Comprehensive OrganicTransformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts,Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons(1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents forOrganic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed.,Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons(1995), and subsequent editions thereof.

Pharmaceutical Compositions

In one embodiment, the invention relates to a pharmaceutical compositioncontaining a modified iRNA agent, as described in the precedingsections, and a pharmaceutically acceptable carrier, as described below.A pharmaceutical composition including the modified iRNA agent is usefulfor treating a disease caused by expression of a target gene. In thisaspect of the invention, the iRNA agent of the invention is formulatedas described below. The pharmaceutical composition is administered in adosage sufficient to inhibit expression of the target gene.

The pharmaceutical compositions of the present invention areadministered in dosages sufficient to inhibit the expression or activityof the target gene. Compositions containing the iRNA agent of theinvention can be administered at surprisingly low dosages. A maximumdosage of 5 mg iRNA agent per kilogram body weight per day may besufficient to inhibit or completely suppress the expression or activityof the target gene.

In general, a suitable dose of modified iRNA agent will be in the rangeof 0.001 to 500 milligrams per kilogram body weight of the recipient perday (e.g., about 1 microgram per kilogram to about 500 milligrams perkilogram, about 100 micrograms per kilogram to about 100 milligrams perkilogram, about 1 milligrams per kilogram to about 75 milligrams perkilogram, about 10 micrograms per kilogram to about 50 milligrams perkilogram, or about 1 microgram per kilogram to about 50 micrograms perkilogram). The pharmaceutical composition may be administered once perday, or the iRNA agent may be administered as two, three, four, five,six or more sub-doses at appropriate intervals throughout the day. Inthat case, the iRNA agent contained in each sub-dose must becorrespondingly smaller in order to achieve the total daily dosage. Thedosage unit can also be compounded for delivery over several days, e.g.,using a conventional sustained release formulation which providessustained release of the iRNA agent over a several day period. Sustainedrelease formulations are well known in the art.

In this embodiment, the dosage unit contains a corresponding multiple ofthe daily dose.

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the infection or disease, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual iRNA agent encompassed by theinvention can be made using conventional methodologies or on the basisof in vivo testing using an appropriate animal model, as describedelsewhere herein.

Advances in mouse genetics have generated a number of mouse models forthe study of various human diseases. For example, mouse repositories canbe found at The Jackson Laboratory, Charles River Laboratories, Taconic,Harlan, Mutant Mouse Regional Resource Centers (MMRRC) National Networkand at the European Mouse Mutant Archive. Such models may be used for invivo testing of iRNA agent, as well as for determining a therapeuticallyeffective dose.

The pharmaceutical compositions encompassed by the invention may beadministered by any means known in the art including, but not limited tooral or parenteral routes, including intravenous, intramuscular,intraperitoneal, subcutaneous, transdermal, airway (aerosol), ocular,rectal, vaginal and topical (including buccal and sublingual)administration. In preferred embodiments, the pharmaceuticalcompositions are administered by intravenous or intraparenteral infusionor injection. The pharmaceutical compositions can also be administeredintraparenchymally, intrathecally, and/or by stereotactic injection.

For oral administration, the iRNA agent useful in the invention willgenerally be provided in the form of tablets or capsules, as a powder orgranules, or as an aqueous solution or suspension.

Tablets for oral use may include the active ingredients mixed withpharmaceutically acceptable excipients such as inert diluents,disintegrating agents, binding agents, lubricating agents, sweeteningagents, flavoring agents, coloring agents and preservatives. Suitableinert diluents include sodium and calcium carbonate, sodium and calciumphosphate, and lactose, while corn starch and alginic acid are suitabledisintegrating agents. Binding agents may include starch and gelatin,while the lubricating agent, if present, will generally be magnesiumstearate, stearic acid or talc. If desired, the tablets may be coatedwith a material such as glyceryl monostearate or glyceryl distearate, todelay absorption in the gastrointestinal tract.

Capsules for oral use include hard gelatin capsules in which the activeingredient is mixed with a solid diluent, and soft gelatin capsuleswherein the active ingredient is mixed with water or an oil such aspeanut oil, liquid paraffin or olive oil.

For intramuscular, intraperitoneal, subcutaneous and intravenous use,the pharmaceutical compositions of the invention will generally beprovided in sterile aqueous solutions or suspensions, buffered to anappropriate pH and isotonicity. Suitable aqueous vehicles includeRinger's solution and isotonic sodium chloride. In a preferredembodiment, the carrier consists exclusively of an aqueous buffer. Inthis context, “exclusively” means no auxiliary agents or encapsulatingsubstances are present which might affect or mediate uptake of iRNAagent in the cells that harbor the target gene or virus. Such substancesinclude, for example, micellar structures, such as liposomes or capsids,as described below. Although microinjection, lipofection, viruses,viroids, capsids, capsoids, or other auxiliary agents are required tointroduce iRNA agent into cell cultures, surprisingly these methods andagents are not necessary for uptake of iRNA agent in vivo. The iRNAagent of the present invention are particularly advantageous in thatthey do not require the use of an auxiliary agent to mediate uptake ofthe iRNA agent into the cell, many of which agents are toxic orassociated with deleterious side effects. Aqueous suspensions accordingto the invention may include suspending agents such as cellulosederivatives, sodium alginate, polyvinyl-pyrrolidone and gum tragacanth,and a wetting agent such as lecithin. Suitable preservatives for aqueoussuspensions include ethyl and n-propyl p-hydroxybenzoate.

The pharmaceutical compositions can also include encapsulatedformulations to protect the iRNA agent against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811; PCT publication WO 91/06309; and European patent publicationEP-A-43075, which are incorporated by reference herein.

Toxicity and therapeutic efficacy of iRNA agent can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.iRNA agents that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosages ofcompositions of the invention are preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anyiRNA agent used in the method of the invention, the therapeuticallyeffective dose can be estimated initially from cell culture assays. Adose may be formulated in animal models to achieve a circulating plasmaconcentration range of the iRNA agent or, when appropriate, of thepolypeptide product of a target sequence (e.g., achieving a decreasedconcentration of the polypeptide) that includes the IC₅₀ (i.e., theconcentration of the test iRNA agent which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

In addition to their administration individually or as a plurality, asdiscussed above, iRNA agents relating to the invention can beadministered in combination with other known agents effective intreating viral infections and diseases. In any event, the administeringphysician can adjust the amount and timing of iRNA agent administrationon the basis of results observed using standard measures of efficacyknown in the art or described herein.

For oral administration, the iRNA agent useful in the invention willgenerally be provided in the form of tablets or capsules, as a powder orgranules, or as an aqueous solution or suspension.

Methods for Treating Diseases Caused by Expression of a Target Gene

In one embodiment, the invention relates to a method for treating asubject having a disease or at risk of developing a disease caused bythe expression of a target gene. In this embodiment, iRNA agents can actas novel therapeutic agents for controlling one or more of cellularproliferative and/or differentiative disorders, disorders associatedwith bone metabolism, immune disorders, hematopoietic disorders,cardiovascular disorders, liver disorders, viral diseases, or metabolicdisorders. The method includes administering a pharmaceuticalcomposition of the invention to the patient (e.g., a human), such thatexpression of the target gene is silenced. Because of their highefficiency and specificity, the iRNA agent of the present inventionspecifically target mRNA of target genes of diseased cells and tissues,as described below, and at surprisingly low dosages. The pharmaceuticalcompositions are formulated as described in the preceding section, whichis hereby incorporated by reference herein.

Examples of genes which can be targeted for treatment include, withoutlimitation, an oncogene (Hanahan, D. and R. A. Weinberg, Cell (2000)100:57; and Yokota, J., Carcinogenesis (2000) 21(3):497-503); a cytokinegene (Rubinstein, M., et al., Cytokine Growth Factor Rev. (1998)9(2):175-81); a idiotype (Id) protein gene (Benezra, R., et al.,Oncogene (2001) 20(58):8334-41; Norton, J. D., J. Cell Sci. (2000)113(22):3897-905); a prion gene (Prusiner, S. B., et al., Cell (1998)93(3):337-48; Safar, J., and S. B. Prusiner, Prog. Brain Res. (1998)117:421-34); a gene that expresses molecules that induce angiogenesis(Gould, V. E. and B. M. Wagner, Hum. Pathol. (2002) 33(11):1061-3);adhesion molecules (Chothia, C. and E. Y. Jones, Annu. Rev. Biochem.(1997) 66:823-62; Parise, L. V., et al., Semin. Cancer Biol. (2000)10(6):407-14); cell surface receptors (Deller, M. C., and Y. E. Jones,Curr. Opin. Struct. Biol. (2000) 10(2):213-9); genes of proteins thatare involved in metastasizing and/or invasive processes (Boyd, D.,Cancer Metastasis Rev. (1996) 15(1):77-89; Yokota, J., Carcinogenesis(2000) 21(3):497-503); genes of proteases as well as of molecules thatregulate apoptosis and the cell cycle (Matrisian, L. M., Curr. Biol.(1999) 9(20):R776-8; Krepela, E., Neoplasma (2001) 48(5):332-49; Basbaumand Werb, Curr. Opin. Cell Biol. (1996) 8:731-738; Birkedal-Hansen, etal., Crit. Rev. Oral Biol. Med. (1993) 4:197-250; Mignatti and Rifkin,Physiol. Rev. (1993) 73:161-195; Stetler-Stevenson, et al., Annu. Rev.Cell Biol. (1993) 9:541-573; Brinkerhoff, E., and L. M. Matrisan, NatureReviews (2002) 3:207-214; Strasser, A., et al., Annu. Rev. Biochem.(2000) 69:217-45; Chao, D. T. and S. J. Korsmeyer, Annu. Rev. Immunol.(1998) 16:395-419; Mullauer, L., et al., Mutat. Res. (2001)488(3):211-31; Fotedar, R., et al., Prog. Cell Cycle Res. (1996)2:147-63; Reed, J. C., Am. J. Pathol. (2000) 157(5):1415-30; D'Ari, R.,Bioassays (2001) 23(7):563-5); genes that express the EGF receptor;Mendelsohn, J. and J. Baselga, Oncogene (2000) 19(56):6550-65; Normanno,N., et al., Front. Biosci. (2001) 6:D685-707); and the multi-drugresistance 1 gene, MDR1 gene (Childs, S., and V. Ling, Imp. Adv. Oncol.(1994) 21-36).

In the prevention of disease, the target gene may be one which isrequired for initiation or maintenance of the disease, or which has beenidentified as being associated with a higher risk of contracting thedisease. In the treatment of disease, the iRNA agent can be brought intocontact with the cells or tissue exhibiting the disease. For example,iRNA agent substantially identical to all or part of a mutated geneassociated with cancer, or one expressed at high levels in tumor cells,may be brought into contact with or introduced into a cancerous cell ortumor gene.

Examples of cellular proliferative and/or differentiative disordersinclude cancer, e.g., a carcinoma, sarcoma, metastatic disorder orhematopoietic neoplastic disorder, such as a leukemia. A metastatictumor can arise from a multitude of primary tumor types, including butnot limited to those of prostate, colon, lung, breast and liver origin.As used herein, the terms “cancer,” “hyperproliferative,” and“neoplastic” refer to cells having the capacity for autonomous growth,i.e., an abnormal state or condition characterized by rapidlyproliferating cell growth. These terms are meant to include all types ofcancerous growths or oncogenic processes, metastatic tissues ormalignantly transformed cells, tissues, or organs, irrespective ofhistopathologic type or stage of invasiveness. Proliferative disordersalso include hematopoietic neoplastic disorders, including diseasesinvolving hyperplastic/neoplastic cells of hematopoietic origin, e.g.,arising from myeloid, lymphoid or erythroid lineages, or precursor cellsthereof.

The pharmaceutical compositions of the present invention can also beused to treat a variety of immune disorders, in particular thoseassociated with overexpression or aberrant expression of a gene orexpression of a mutant gene. Examples of hematopoietic disorders ordiseases include, without limitation, autoimmune diseases (including,for example, diabetes mellitus, arthritis (including rheumatoidarthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriaticarthritis), multiple sclerosis, encephalomyelitis, myasthenia gravis,systemic lupus erythematosis, automimmune thyroiditis, dermatitis(including atopic dermatitis and eczematous dermatitis), psoriasis,Sjogren's Syndrome, Crohn's disease, aphthous ulcer, iritis,conjunctivitis, keratoconjunctivitis, ulcerative colitis, asthma,allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis,proctitis, drug eruptions, leprosy reversal reactions, erythema nodosumleprosum, autoimmune uveitis, allergic encephalomyelitis, acutenecrotizing hemorrhagic encephalopathy, idiopathic bilateral progressivesensorineural hearing, loss, aplastic anemia, pure red cell anemia,idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis,chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue,lichen planus, Graves' disease, sarcoidosis, primary biliary cirrhosis,uveitis posterior, and interstitial lung fibrosis), graft-versus-hostdisease, cases of transplantation, and allergy.

In another embodiment, the invention relates to methods for treatingviral diseases, including but not limited to hepatitis C, hepatitis B,herpes simplex virus (HSV), HIV-AIDS, poliovirus, and smallpox virus.iRNA agent of the invention are prepared as described herein to targetexpressed sequences of a virus, thus ameliorating viral activity andreplication. The iRNA agents can be used in the treatment and/ordiagnosis of viral infected tissue, both animal and plant. Also, suchiRNA agent can be used in the treatment of virus-associated carcinoma,such as hepatocellular cancer.

For example, the iRNA agent of the present invention are useful fortreating a subject having an infection or a disease associated with thereplication or activity of a (+) strand RNA virus having a 3′-UTR, suchas HCV. In this embodiment, the iRNA agent can act as novel therapeuticagents for inhibiting replication of the virus. The method includesadministering a pharmaceutical composition of the invention to thepatient (e.g., a human), such that viral replication is inhibited.Examples of (+) strand RNA viruses which can be targeted for inhibitioninclude, without limitation, picornaviruses, caliciviruses, nodaviruses,coronaviruses, arteriviruses, flaviviruses, and togaviruses. Examples ofpicornaviruses include enterovirus (poliovirus 1), rhinovirus (humanrhinovirus 1A), hepatovirus (hepatitis A virus), cardiovirus(encephalomyocarditis virus), aphthovirus (foot-and-mouth disease virusO), and parechovirus (human echovirus 22). Examples of calicivirusesinclude vesiculovirus (swine vesicular exanthema virus), lagovirus(rabbit hemorrhagic disease virus), “Norwalk-like viruses” (Norwalkvirus), “Sapporo-like viruses” (Sapporo virus), and “hepatitis E-likeviruses” (hepatitis E virus). Betanodavirus (striped jack nervousnecrosis virus) is the representative nodavirus. Coronaviruses includecoronavirus (avian infections bronchitis virus) and torovirus (Bernevirus). Arterivirus (equine arteritis virus) is the representativearteriviridus. Togavirises include alphavirus (Sindbis virus) andrubivirus (Rubella virus). Finally, the flaviviruses include flavivirus(Yellow fever virus), pestivirus (bovine diarrhea virus), andhepacivirus (hepatitis C virus). In a preferred embodiment, the virus ishepacivirus, the hepatitis C virus. Although the foregoing listexemplifies vertebrate viruses, the present invention encompasses thecompositions and methods for treating infections and diseases caused byany (+) strand RNA virus having a 3′-UTR, regardless of the host. Forexample, the invention encompasses the treatment of plant diseasescaused by sequiviruses, comoviruses, potyviruses, sobemovirus,luteoviruses, tombusviruses, tobavirus, tobravirus, bromoviruses, andclosteroviruses.

The pharmaceutical compositions encompassed by the invention may beadministered by any means known in the art including, but not limitedto, oral or parenteral routes, including intravenous, intramuscular,intraperitoneal, subcutaneous, transdermal, airway (aerosol), ocular,rectal, vaginal, and topical (including buccal and sublingual)administration. In preferred embodiments, the pharmaceuticalcompositions are administered by intravenous or intraparenteral infusionor injection. The pharmaceutical compositions can also be administeredintraparenchymally, intrathecally, and/or by stereotactic injection.

Methods for Inhibiting Expression of a Target Gene.

In yet another aspect, the invention relates to a method for inhibitingthe expression of a target gene in a cell or organism. In oneembodiment, the method includes administering the inventive iRNA agentor a pharmaceutical composition containing the iRNA agent to a cell oran organism, such as a mammal, such that expression of the target geneis silenced. Because of their surprisingly improved stability andbioavailability, the iRNA agent of the present invention effectivelyinhibit expression or activity of target genes at surprisingly lowdosages. Compositions and methods for inhibiting the expression of atarget gene using iRNA agent can be performed as described in thepreceding sections, particularly Sections 4 and 5.

In this embodiment, a pharmaceutical composition containing the iRNAagent may be administered by any means known in the art including, butnot limited to oral or parenteral routes, including intravenous,intramuscular, intraperitoneal, subcutaneous, transdermal, airway(aerosol), ocular, rectal, vaginal, and topical (including buccal andsublingual) administration. In preferred embodiments, the pharmaceuticalcompositions are administered by intravenous or intraparenteral infusionor injection. The pharmaceutical compositions can also be administeredintraparenchymally, intrathecally, and/or by stereotactic injection.

The methods for inhibiting the expression of a target gene can beapplied to any gene one wishes to silence, thereby specificallyinhibiting its expression, provided the cell or organism in which thetarget gene is expressed includes the cellular machinery which effectsRNA interference. Examples of genes which can be targeted for silencinginclude, without limitation, developmental genes including but notlimited to adhesion molecules, cyclin kinase inhibitors, Wnt familymembers, Pax family members, Winged helix family members, Hox familymembers, cytokines/lymphokines and their receptors,growth/differentiation factors and their receptors, andneurotransmitters and their receptors; (2) oncogenes including but notlimited to ABLI, BCL1, BCL2, BCL6, CBFA2, CBL, CSFIR, ERBA, ERBB, EBRB2,ETS1, ETS1, ETV6, FGR, FOS, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2,MLL, MYB, MYC, MYCL1, MYCN, NRAS, PIM1, PML, RET, SRC, TALl, TCL3 andYES; (3) tumor suppresser genes including but not limited to APC, BRCA1,BRCA2, MADH4, MCC, NF1, NF2, RB1, TP53 and WT 1; and (4) enzymesincluding but not limited to ACP desaturases and hydroxylases,ADP-glucose pyrophorylases, ATPases, alcohol dehydrogenases, amylases,amyloglucosidases, catalases, cellulases, cyclooxygenases,decarboxylases, dextrinases, DNA and RNA polymerases, galactosidases,glucanases, glucose oxidases, GTPases, helicases, hemicellulases,integrases, invertases, isomerases, kinases, lactases, lipases,lipoxygenases, lysozymes, pectinesterases, peroxidases, phosphatases,phospholipases, phosphorylases, polygalacturonases, proteinases andpeptideases, pullanases, recombinases, reverse transcriptases,topoisomerases, and xylanases.

In addition to in vivo gene inhibition, the skilled artisan willappreciate that the iRNA agent of the present invention are useful in awide variety of in vitro applications. Such in vitro applications,include, for example, scientific and commercial research (e.g.,elucidation of physiological pathways, drug discovery and development),and medical and veterinary diagnostics. In general, the method involvesthe introduction of the iRNA agent into a cell using known techniques(e.g., absorption through cellular processes, or by auxiliary agents ordevices, such as electroporation and lipofection), then maintaining thecell for a time sufficient to obtain degradation of an mRNA transcriptof the target gene.

In one aspect the invention provides a method of modulating theexpression of a target gene in a cell, comprising providing to said cellan iRNA agent of this invention.

In one embodiment, the target gene is selected from the group consistingof Factor VII, Eg5, PCSK9, TPX2, apoB, SAA, TTR, RSV, PDGF beta gene,Erb-B gene, Src gene, CRK gene, GRB2 gene, RAS gene, MEKK gene, JNKgene, RAF gene, Erk1/2 gene, PCNA(p21) gene, MYB gene, JUN gene, FOSgene, BCL-2 gene, Cyclin D gene, VEGF gene, EGFR gene, Cyclin A gene,Cyclin E gene, WNT-1 gene, beta-catenin gene, c-MET gene, PKC gene, NFKBgene, STAT3 gene, survivin gene, Her2/Neu gene, topoisomerase I gene,topoisomerase II alpha gene, mutations in the p73 gene, mutations in thep21(WAF1/CIP1) gene, mutations in the p27(KIP1) gene, mutations in thePPM1D gene, mutations in the RAS gene, mutations in the caveolin I gene,mutations in the MIB I gene, mutations in the MTAI gene, mutations inthe M68 gene, mutations in tumor suppressor genes, and mutations in thep53 tumor suppressor gene.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

EXAMPLES Example 1 Synthesis of Folate Conjugates 108 and 109

In order to conjugate the Folic acid to the siRNA the following strategywas used. Folic acid was conjugated to the 3′ end using the solidsupport, 108 and to the 5′ end using the amidite 109 as follows.

Preparation of 102:

Glutamate 100 (2.05 g, 6.95 mmol) and amine 101 (4.50 g, 6.95 mmol) wastaken together in a mixture of DCM/DMF (50 mL, 2:1). To this reactionmixture HBTU (3.42 g, 1.3 eq.) and DIEA (2.43 ml, 2 eq.) was added andstirred for 30 minutes at ambient temperature. The progress of thereaction was monitored by TLC (Ethyl acetate); solvents are removedunder reduced pressure and the residue was extracted withdichloromethane, washed with water (2 times) and brine. The organiclayer was dried over anhydrous sodium sulfate. Solvents were removed andresidue was purified by chromatography (during the packing of columnplease add few drops of TEA, First elute with 1:1 ethyl acetate/hexane,followed ethyl acetate) to get the product 102 as yellow gum (Yield,6.35 g, 97%). ¹H NMR (DMSO-d₆, 400 MHz) δ=7.94 (bs, 1H), 7.70-7.77 (m,1H), 7.15-7.40 (m, 14H), 6.80-6.90 (m, 4H), 5.01 (s, 2H), 4.69-4.50 (m,1H), 4.01-4.15 (m, 1H), 3.71 (s, 6H), 3.61 (s, 3H), 2.85-3.40 (m, 5H),2.46-2.52 (m, 1H), 1.80-2.22 (m, 7H), 0.89-1.50 (m, 6H), 0.83 (s, 9H),0.04-0.01 (m, 6H). ¹³C NMR DMSO-d₆) δ=172.68, 170.91, 170.82, 170.80,170.28, 164.56, 162.25, 158.11, 157.99, 156.05, 145.06, 144.74, 136.86,135.77, 135.59, 135.33, 129. 57, 128.89, 128.31, 127.78, 127.74, 127.69,127.62, 127.51, 126.58, 113.08, 112.71, 85.49, 85.24, 70.32, 65.47,63.36, 59.71, 54.99, 54.96, 54.86, 54.60, 53.48, 51.78, 38.33, 38.20,36.78, 35.73, 33.93, 31.45, 30.71, 29.02, 26.65, 26.65, 26.14, 25.62,24.12, 20.70, 17.65, 17.62, 14.04, −4.91, −4.93, −4.97. MS. Molecularweight calculated for C₅₂H₆₇N₃O₉Si, Cal. 906.19. Found 907.2 (MH⁺).

Preparation of 103:

The Cbz protected amine 102 (6.30 g 6.80 mmol) was dissolved in amixture of MeOH/Ethyl acetate (1:3, 75 mL) and degassed with argon. Pd/C(0.80 g, 10 wt % Degussa wet type) was added and degassed and purgedwith hydrogen. The mixture was stirred under hydrogen (Balloon pressure)for 6-9 hrs. Reaction was monitored by TLC (Ethyl acetate, PMA stain).The TLC of the reaction mixture showed the complete disappearance of thestarting Cbz protected amine. The reaction mixture was filtered througha pad of celite, washed with a mixture of MeOH/EtOAc (100 mL) and thecombined filtrates were concentrated. The residue was dried under highvacuum overnight to provide the product 103 as pale yellow foam (5.30 g,98%). It was directly used for the next reaction with out furtherpurification. ¹H NMR (DMSO-d₆, 400 MHz) δ=7.94 (bs, 1H), 7.70-7.75 (m,1H), 7.12-7.33 (m, 9H), 6.81-6.88 (m, 4H), 4.50-4.64 (m, 1H), 4.05-4.17(m, 1H), 3.71 (m, 6H), 3.59 (s, 3H), 3.19-3.40 (m, 2H), 2.85-3.05 (m,2H), 2.47-2.52 (m, 1H), 1.80-2.22 (m, 7H), 0.89-1.50 (m, 6H), 0.83 (s,9H), 0.04-0.01 (m, 6H). ¹³C NMR DMSO-d₆) δ=176.23, 176.11, 171.74,171.53, 170.98, 165.15, 162.96, 158.46, 158.33, 158.15, 145.37, 145.04,140.04, 136.10, 135.94, 135.74, 135.64, 129.92, 129.92, 128.55, 128.24,128.13, 127.83, 127.17, 127.01, 113.52, 113.42, 113.09, 86.25, 85.62,70.67, 69.67, 65.50, 63.65, 60.22, 56.06, 55.44, 55.35, 55.32, 55.06,53.96, 53.55, 51.86, 37.13, 36.22, 34.31, 32.78, 31.93, 31.17, 30.67,29.21, 26.42, 25.94, 24.84, 24.46, 21.04, 17.81, 17.95, 14.35, −4.60,−4.65 MS. Molecular weight calculated for C₄₄H₆₃F₃N₃O₈Si, Cal. 789.44.Found 790.5 (MH⁺).

Preparation of 105:

N¹⁰-(Trifluoroacetyl)pteroic acid 104 (2.30 g, 6.00 mmol) and amine 103(5.75 g, 1.2 eq) were dissolved in DMF (60 mL, it takes about 15-20minutes to dissolve the compounds in solution). HBTU (3.01 g, 1.3 eq.)followed by DIEA (3 mL, 3 eq.) were added and stirred for 30 minutes.Reaction was monitored by TLC (8% MeOH/DCM, PMA stain). TLC of thereaction mixture showed completion of reaction. Solvents were removedunder reduced pressure and the residue extracted with DCM, washed withwater and brine. The organic layer was dried over anhydrous sodiumsulfate. Solvents were removed and residue was purified bychromatography (3-8% MeOH/DCM) to get the product 105 a pale yellowsolid (Yield=6.77 g, 93%). ¹H NMR (DMSO-d₆, 400 MHz) δ=8.88-8.90 (d,J=7.07 Hz, 1H), 8.60 (bs, 1H), 7.90 (d, J=8.4 Hz, 2H), 7.80 (t, J=8.4Hz, 1H), 7.60 (d, J=8.06 Hz, 2H), 7.12-7.30 (m, 9H), 6.80-6.89 (m, 4H),5.09 (bs, 2H), 4.52-4.68 (m, 1H), 4.32-4.40 (m, 1H), 4.05-1.12 (m, 1H),3.71 (s, 6H), 3.62 (s, 3H), 2.86-3.30 (m, 6H), 1.75-2.23 (m, 8H),1.10-145 (m, 6H), 0.82 (s, 9H), 0.04-0.01 (m, 6H). ¹³C NMR DMSO-d₆)δ=172.38, 170.96, 170.88, 165.60, 158.11, 157.99, 145.08, 141.66,135.77, 135.60, 134.19, 129.59, 128.74, 128.74, 128.45, 128.13, 127.78,127.52, 113.20, 113.11, 85.24, 55.01, 54.98, 54.91, 52.46, 51.90, 45.72,40.12, 39.92, 39.71, 39.50, 39.29, 39.08, 38.87, 25.65, 17.69, 8.68,−4.88, −4.94. MS. Molecular weight calculated for C₆₀H₇₂F₃N₉O₁₁Si, Cal.1179.51. Found 1181.0 (MH⁺).

Preparation of 106:

To a solution of 105 (6.75 g, 5.72 mmol) in a mixture of DCM/Pyridine(100 mL, 1:1) DMAP (1.00 g, 1. 5 eq.) was added and cooled in anice-water bath. Isobutyric anhydride (10 mL, excess) was added andstirred overnight. The reaction was monitored by TLC. Reaction mixturewas quenched with MeOH. Solvents were removed and the residue extractedwith DCM, washed with water and brine; dried over anhydrous sodiumsulfate. Solvents were removed and the residue was purified bychromatography (first ethyl acetate then 3-5% MeOH/DCM) to get theproduct 106 as pale yellow solid (Yield=6.70 g, 94%). ¹H NMR (DMSO-d₆,400 MHz) δ=11.90-12.32 (m, 3H), 8.84-8.90 (m, 2H), 7.96-7.90 (m, 3H),7.60-7.84 (m, 3H), 7.10-7.35 (m, 9H), 6.80-6.90 (m, 4H), 5.20 (bs, 2H),4.50-4.67 (m, 1H), 4.34-4.42 (m, 1H), 4.05-4.12 (m, 1H), 3.71 (s, 6H),3.61 (s, 3H), 2.86-3.30 (m, 6H), 1.75-2.23 (m, 6H), 1.17 (d, J=7.08 Hz,3H), 1.05 (d, J=7.08 Hz, 3H), 1.10-1.45 (m, 6H), 0.82 (s, 9H), 0.04-0.01(m, 6H). ¹³C NMR DMSO-d₆) δ=180.81, 177.83, 172.37, 170.98, 170.94,170.87, 165.56, 162.28, 159.89, 158.12, 157.99, 155.90, 155.55, 149.99,149.87, 147.79, 145.10, 144.78, 141.79, 135.77, 135.61, 135.44, 135.35,134.25, 130.55, 129.60, 128.71, 128.51, 127.88, 127.78, 127.53, 126.60,117.54, 114.66, 113.19, 113.09, 85.86, 85.25, 70.35, 63.37, 59.75,55.00, 54.97, 54.62, 54.02, 52.49, 51.89, 45.75, 38.37, 38.22, 36.79,35.76, 35.00, 33.94, 33.12, 31.69, 30.74, 29.04, 26.30, 26.16, 25.63,24.14, 20.74, 18.89, 18.74, 17.68, 17.65, 14.07, 8.67, −4.89, −4.96. MS.Molecular weight calculated for C₆₄H₇₈F₃N₉O₁₂Si, Cal. 1249.55. Found:1248.4 (M−H)⁻.

Preparation of 107:

Compound 106 (6.70 g, 5.36 mmol) was dissolved in acetonitrile (50 mL)and Triethylamine (20 mL). To that HF/TEA (20 mL) was added and stirredfor 3 hrs. The reaction was monitored by TLC (5% MeOH/DCM). TLC showedcomplete disappearance of starting material. Water and sodiumbicarbonate solution was added to the reaction mixture and extractedwith DCM. The organic layer was dried over sodium sulfate. Solvents wereremoved and the residue was purified by chromatography (2-5% MeOH/DCM)to get the product 107 as pale yellow solid (Yield=5.10 g, 84%). ¹H NMR(DMSO-d₆, 400 MHz) δ=11.45-11.95 (m, 2H), 8.81-8.95 (m, 2H), 7.60-7.94(m, 5H), 7.10-7.36 (m, 9H), 6.78-6.91 (m, 4H), 5.19 (bs, 2H), 4.85-5.01(bs, 1H), 4.34-4.42 (m, 1H), 3.71 (s, 6H), 3.61 (s, 3H), 2.86-3.55 (m,9H), 1.75-2.23 (m, 6H), 1.16 (d, J=7.08 Hz, 3H), 1.10-1.45 (m, 6H), 0.95(d, J=7.08 Hz, 3H). ¹³C NMR DMSO-d₆) δ=180.36, 172.39, 170.98, 170.87,170.34, 165.60, 160.60, 158.11, 157.98, 155.89, 155.54, 154.97, 151.03,149.68, 147.30, 145.10, 144.76, 141.76, 135.88, 135.74, 135.49, 135.44,134.24, 130.50, 129.62, 129.56, 128.74, 128.50, 127.88, 127.78, 127.59,126.59, 117.54, 114.67, 113.19, 113.10, 85.78, 85.11, 68.59, 67.47,65.18, 63.34, 59.76, 55.01, 54.98, 52.48, 51.90, 38.38, 36.29, 34.91,34.15, 32.51, 31.67, 29.05, 26.29, 26.19, 24.18, 20.75, 18.84, 14.08,11.07. MS. Molecular weight calculated for C₅₈H₆₄F₃N₉O₁₂, Cal. 1135.46.Found 1135.0 (M−H)⁻.

Preparation of Long alkyl chain CPG 108:

Hydroxy derivative 107 (0.500 g, 0.441 mmol) was dissolved in DCM (10mL) to that Succinic anhydride (0.088 g, 2 eq) and DMAP (0.160 g, 3 eq.)were added and stirred overnight. TLC showed completion of reaction. Thereaction mixture was diluted with DCM (20 mL), washed successively withcold dilute citric acid and water (2 times), dried over sodium sulfate.Solvents were removed and dried under high vacuum to get the succinate.PPh₃ (0.150 g, 1.3 eq.), DMAP (0.080 g, 1.5 eq.) and the succinate fromthe previous step were dissolved in a mixture of acetonitrile and DCM (6mL). A solution of DTNP (0.143 g, 1.05 eq.) in DCM (1 mL) was added tothe above solution. The mixture was slowly shaken for 3-4 minutes. Longchain alkyl amine-CPG (lcaa CPG, 2.05 g, 133 μmol/g) was added to themixture and gently shaken for 2 h. The CPG was filtered, successivelywashed with DCM, mixture of MeOH/DCM (1:9) and DCM until filtrateremained colorless and dried. The dried CPG was transferred into anotherflask treated with Ac₂O in pyridine (25%) in the presence of TEA (1 mL)for 15 min. under gentle shaking. Finally the CPG was filtered, washedwith DCM, DCM:MeOH (9:1), followed by DCM and ether. The CPG 108 wasdried under vacuum overnight and the loading was measured as reported(2.14 g, loading 53.3 μmol/g).

Preparation of Phosphoramidite 109:

To a solution of 107 (0.20 g, 0.176 mmol) in EtOAc/DCM (4:1, 10 mL)chlorophosphonate reagent (0.086 mL, 2 eq.) was added, followed by TEA(0.53 mL, 2 eq.). After 10 minutes the solution becomes cloudy. Thereaction was monitored by TLC. TLC showed reaction was complete in 30minutes, diluted with DCM washed with sodium bicarbonate solution anddried over sodium sulfate. Solvents were removed and the residue wasdissolved small amount of DCM/EtOAc mixture and precipitated the productwith hexane (Yield=195 mg, 82%). MS. Molecular weight calculated forC₆₇H₈₁F₃N₁₁O₁₃P, Cal. 1335.57. Found. 1136.60 (M+H).

Example 2 Synthesis of Pteroic Acid Precursor 110

In another method the appropriately substituted pteroic acid precursor110, amenable for RNA synthesis was prepared as follows.

Synthesis of4-[(2-isobutyrylamino-4-oxo-3,4-dihydro-pteridin-6-ylmethyl)-(2,2,2-trifluoroacetyl)-amino]benzoicacid 110

To a suspension of pteroic acid (25 g, 61.2 mmol) and DMAP (11.25 g, 92mmol) in anhydrous pyridine (400 mL), TBDPS chloride (42 g, 153 mmol)was added. The reaction mixture was stirred at room temperature for 30 hafter which isobutric anhydride (14.6 g, 92 mmol) was added and themixture was slightly warmed. An additional 60 mL of pyridine was alsoadded and the reaction mixture was stirred at room temperatureovernight. The reaction mixture became homogenous after which pyridineand other volatiles were concentrated in a rotary evaporator. Theresidue was stirred with EtOAc (1 L) and acetic acid (100 mL) and water(500 mL) for 24 h. The thus obtained slurry was filtered, the residuewas washed with water (500 mL), EtOAc (1 L) and dried to obtain the pureproduct as a white solid (26.1 g, 89%). ¹H NMR (DMSO-d₆, 400 MHz) δ=8.87(s, 1H), 7.95 (d, J=8.6 Hz, 2H), 7.67 (d, J=8.6 Hz, 2H), 5.21 (s, 2H),2.79-2.74 (m, 1H), 1.12 (d, J=6.83 Hz, 6H), ¹³C NMR (DMSO-d₆) δ=180.72,166.49, 159.25, 149.87, 147.68, 142.69, 136.34, 134.45, 130.54, 129.16,128.86, 127.49, 34.96, 33.09, 26.52, 18.88, 18.74. ¹⁹F NMR (DMSO-d₆)δ−64.32. MS. Molecular weight calculated for C₂₀H₁₇F₃N₆O₅, Cal. 478.12.Found 479.12 (MH⁺).

Example 3 Synthesis of Disulfide Precursor 112

In order to conjugate Folic acid to siRNA via a cleavable disulfidelinker the following strategy was used. The pyridyl disulfide precursor112 was synthesized by conjugating hydroxyprolinol derivative 101 withthe NHS ester 111 as follows.

Synthesis of 4-methyl-4-(pyridine-2-yldisulfanyl)-pentanoicacid{6-[2-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-(tert-butyl-dimethyl-silanyloxy)-pyrrolidin-1-yl]-6-oxo-hexyl}-amide112

To a solution of the amine 101 (3.23 g, 5 mmol) in dichloromethane (50mL), diisopropylethylamine (1.27 g, 10 mmol) and the NHS ester 111 (1.8g, 5 mmol) was added and the mixture was stirred at room temperatureovernight. The reaction mixture was diluted with CH₂Cl₂ (200 mL) and theorganic layer was washed with satd. NaHCO₃ (100 mL), water (100 mL),brine (100 mL) and dried (Na₂SO₄). Solvents were removed and the residuewas purified by chromatography (2-5% MeOH/DCM) to get the product 112 aspale yellow foam (Yield=4.10 g, 92%). ¹H NMR (DMSO-d₆, 400 MHz) δ=8.39(d, J=4.6 Hz, 1H), 7.80-7.73 (m, 3H), 7.33-7.24 (m, 4H), 7.22-7.16 (m,5H), 6.88-6.84 (m, 4H), 4.66-4.62 (m, 0.7H), 4.58-4.50 (m, 0.3H),4.16-4.06 (m, 1H), 3.78-3.66 (m, 2H), 3.26-3.18 (m, 2H), 3.12-2.89 (m,4H), 2.24-2.12 (m, 4H), 2.02-1.86 (m, 1H), 1.84-1.76 (m, 3H), 1.50-1.10(m, 6H), 1.20 (s, 3H), 0.83 (s, 9H), 0.81 (s, 3H), 0.04-0.01 (m, 6H).¹³C NMR (DMSO-d₆) δ=171.23, 170.91, 170.81, 160.02, 158.09, 157.96,149.14, 145.05, 144.73, 137.51, 135.75, 135.58, 135.42, 135.32, 129.56,127.85, 127.75, 127.50, 126.73, 126.58, 120.98, 119.22, 113.18, 113.09,85.83, 85.23, 70.31, 69.31, 65.17, 63.34, 54.99, 54.96, 54.60, 52.19,38.30, 36.77, 36.43, 33.92, 32.41, 31.01, 28.94, 26.87, 26.11, 25.64,24.08, 17.66, −4.88, −4.91, −4.94. MS. Molecular weight calculated forC₄₉H₆₇N₃O₆S₂Si, Cal. 885.42. Found 886.42 (MH⁺).

Example 4 Synthesis of Folate Conjugates 121 and 122

The pyridyl disulfide 112 is treated with the thiol 114 to get thedisulfide 115 which on treatment with the glutamate 116 provided theamide 117. The Fmoc disulfide 117 is treated with piperidine to removethe Fmoc protecting group to give the amine 118 which on treatment withthe protected pteroic acid 110 provided the couple product 119. TheTBDMS protecting group in 119 is removed using HF:NEt₃ complex toprovide the hydroxyprolinol precursor 120. The hydroxyprolinol 120 isconverted to the solid support 122 via the succinate and the amidite 121as follows.

Example 5 Synthesis of Disulfide Precursor 124

In another example the folate conjugate without the stabilizing gemdimethyl group next to the disulfide link was probed. In a proceduresimilar to that described above for compound 112 (example 3) is used tosynthesize 124 as follows.

Example 6 Synthesis of Folate Conjugates 130 and 131

In another approach the Folic acid was conjugated to the siRNA via adisulfide link through cystine as follows.

Example 7

In another embodiment the disulfide linkage was placed closer to thesiRNA as follows.

Example 8 Synthesis of Compounds Folic Acid Precursors Amenable toOligonucleotide Synthesis

In order to synthesize an appropriately substituted more versatileprecursor of Folic acid amenable for RNA synthesis, the followingstrategy was used. In this method the protected Folic acid 110 wastreated with the γ-tert-butyl, α-Me ester of glutamic acid, 142 toobtain the ester 143 which on treatment with TFA/CH₂Cl₂ provided theprecursor 144. The precursor 144 was coupled with various amines like145, 151, 155, 161, 165, 172 and 176 provided the couple products 146,152, 156, 162, 166, 173 and 177 respectively. These products are thenconverted to solid-supports 147, 153, 157, 163, 167, 174 and 178, andphosphoramidites 148, 154, 158, 164, 168 and 175.

Synthesis of 147

Synthesis of2-{4-[(2-isobutyrylamino-4-oxo-3,4-dihydro-pteridin-6-ylmethyl)-(2,2,2-trifluoroacetyl)-amino}-pentanedioicacid 5-tert-butyl ester 1-methyl ester 143

In a representative procedure, the pteroic acid precursor 110 (2.4 g, 5mmol) was dissolved in anhydrous DMF (20 mL), HBTU (1.9 g, 1 eq.)followed by DIEA (1 mL, 5 eq.) were added and stirred for 20 minutes. Tothis reaction mixture the amine hydrochloride 142 (1.2 g, 1 eq) wasadded as a solution in DMF (6 mL). Reaction was monitored by TLC (8%MeOH/DCM, PMA stain). TLC of the reaction mixture showed completion ofthe reaction. The reaction mixture was slowly poured in ice withvigorous stirring. The precipitated product was filtered to get theproduct 143 as a white solid (Yield=2.85 g, 86%). ¹H NMR (DMSO-d₆, 400MHz) δ=12.33 (s, 1H), 11.94 (s, 1H), 8.88 (s, 1H), 8.82 (d, J=7.3 Hz,1H), 7.90 (d, J=8.6 Hz, 2H), 7.68 (d, J=8.4 Hz, 2H), 5.22 (s, 2H),4.46-4.40 (m, 1H), 3.62 (s, 3H), 2.86-2.73 (m, 1H), 2.32 (t, J=7.4 Hz,2H) 2.05-1.90 (m, 2H), 1.35 (m, 9H), 1.12 (d, J=6.8 Hz, 6H). ¹³C NMRDMSO-d₆) δ=180.75, 172.13, 171.45, 165.64, 159.10, 154.80, 149.97,149.79, 147.72, 141.75, 134.15, 130.53, 128.70, 128.49, 117.50, 114.64,79.79, 51.96, 51.91, 34.96, 31.22, 27.68, 25.71, 18.72. MS. Molecularweight calculated for C₃₀H₃₄F₃N₇O₈, Cal. 677.63. Found 676.72 (M−H⁻).

Synthesis of2-{4-[(2-isobutyrylamino-4-oxo-3,4-dihydro-pteridin-6-ylmethyl)-(2,2,2-trifluoroacetyl)-amino}-pentanedioicacid 1-methyl ester 144

The ester 143 (2 g, 2. 9 mmol) was dissolved in 20 mL of 50% TFA indichloromethane and the solution was stirred at room temperature for 30min. after which the TLC showed the complete disappearance of thestarting ester. The reaction mixture was concentrated and the residuewas crystallized from CH₂Cl₂:Hexanes (2:3) and crystallized product wasfiltered off and dried to obtain the pure product 144 (1.76 g, 96%) asoff white powder. ¹H NMR (DMSO-d₆, 400 MHz) δ=12.32 (bs, 1H), 11.94 (s,1H), 8.88 (s, 1H), 8.84 (d, J=7.4 Hz, 1H), 7.90 (d, J=8.3 Hz, 2H), 7.69(d, J=8.3 Hz, 2H), 5.22 (s, 2H), 4.45-4.41 (m, 1H), 3.62 (s, 3H),2.78-2.75 (m, 1H), 2.35 (t, J=7.4 Hz, 2H) 2.07-1.92 (m, 2H), 1.12 (d,J=6.8 Hz, 6H). ¹³C NMR DMSO-d₆) δ=180.77, 173.70, 172.19, 165.70,159.21, 155.54, 149.93, 149.84, 147.75, 141.78, 134.18, 130.53, 128.71,128.49, 117.51, 114.64, 53.98, 52.06, 51.93, 34.97, 30.11, 25.68, 18.73.MS. Molecular weight calculated for C₂₆H₂₆F₃N₇O₈, Cal. 621.18. Found620.18 (M−H⁻).

Preparation of 146:

N¹⁰-(Trifluoroacetyl)pteroic acid 144 (2.30 g, 6.00 mmol) is dissolvedin DMF (60 mL, it takes about 15-20 minutes to dissolve the compounds insolution). HBTU (3.01 g, 1.3 eq.) followed by DIEA (3 mL, 3 eq.) areadded and stirred for 30 minutes then the amine 145 (5.75 g, 1.2 eq) isadded and the reaction mixture is stirred at room temperature. Reactionis monitored by TLC (8% MeOH/DCM, PMA stain). TLC of the reactionmixture showed the completion of reaction. Solvents are removed underreduced pressure and the residue extracted with DCM, washed with waterand brine. The organic layer is dried over anhydrous sodium sulfate.Solvents are removed and residue was purified by chromatography (3-8%MeOH/DCM) to get the product 146 as a foam (Yield=6.77 g, 93%). ¹H NMR(DMSO-d₆, 400 MHz) δ=8.88-8.90 (d, J=7.07 Hz, 1H), 8.60 (bs, 1H), 7.90(d, J=8.4 Hz, 2H), 7.80 (t, J=8.4 Hz, 1H), 7.60 (d, J=8.06 Hz, 2H),7.12-7.30 (m, 9H), 6.80-6.89 (m, 4H), 5.09 (bs, 2H), 4.52-4.68 (m, 1H),4.32-4.40 (m, 1H), 4.05-1.12 (m, 1H), 3.71 (s, 6H), 3.62 (s, 3H),2.86-3.30 (m, 6H), 1.75-2.23 (m, 8H), 1.10-145 (m, 6H), 0.82 (s, 9H),0.04-0.01 (m, 6H). ¹³C NMR DMSO-d₆) δ=172.38, 170.96, 170.88, 165.60,158.11, 157.99, 145.08, 141.66, 135.77, 135.60, 134.19, 129.59, 128.74,128.74, 128.45, 128.13, 127.78, 127.52, 113.20, 113.11, 85.24, 55.01,54.98, 54.91, 52.46, 51.90, 45.72, 40.12, 39.92, 39.71, 39.50, 39.29,39.08, 38.87, 25.65, 17.69, 8.68, −4.88, −4.94. MS. Molecular weightcalculated for C₆₀H₆₈F₃N₉O₁₂S₂, Cal. 1228.36. Found 1227.3 (M−H)⁻.

Preparation of Long alkyl chain CPG 147:

Hydroxy derivative 146 (0.7 g, 0.441 mmol) was dissolved in DCM (10 mL)to that Succinic anhydride (0.088 g, 2 eq) and DMAP (0.160 g, 3 eq.)were added and stirred overnight. TLC showed completion of reaction. Thereaction mixture was diluted with DCM (20 mL), washed successively withcold dilute citric acid and water (2 times), dried over sodium sulfate.Solvents were removed and dried under high vacuum to get the succinate.PPh₃ (0.150 g, 1.3 eq.), DMAP (0.080 g, 1.5 eq.) and the succinate fromthe previous step were dissolved in a mixture of acetonitrile and DCM (6mL). A solution of DTNP (0.143 g, 1.05 eq.) in DCM (1 mL) was added tothe above solution. The mixture was slowly shaken for 3-4 minutes. Longchain alkyl amine-Polystyrene (5.05 g, 250 mol/g) was added to themixture and gently shaken for 2 h. The CPG was filtered, successivelywashed with DCM, mixture of MeOH/DCM (1:9) and DCM until filtrateremained colorless and dried. The dried support was transferred intoanother flask treated with Ac₂O in pyridine (25%) in the presence of TEA(1 mL) for 15 min. under gentle shaking. Finally the CPG was filtered,washed with DCM, DCM:MeOH (9:1), followed by DCM and ether. The support147 was dried under vacuum overnight and the loading was measured asreported (5.14 g, loading 78 μmol/g).

Synthesis of Compound 153

Using a similar procedure to that described above, a folate conjugatedsolid support with a cleavable disulfide linkage was synthesized asfollows. Treatment of the protected folic acid 144 with the amine 151provided the coupled precursor 152 as white foam. The amine 151 wasprepared by coupling the amine 145 with the azido acid 149 followed bythe reduction of the azide group using triphenyl phosphine in thepresence of water.

Synthesis of Azide 150:

Using a similar procedure to that used for the synthesis of 146,coupling of the amine 145 (2 g, 3.2 mmol) with the azido acid 149 (0.9g, 3.2 mmol) provided the coupled azide 150 (1.86 g, 64%) as a foam. ¹HNMR (DMSO-d₆, 400 MHz) δ=7.94-7.90 (m, 2H), 7.30-7.25 (m, 6H), 7.24-7.14(m, 7H), 6.87-6.84 (m, 6H), 5.22 (d, 0.7H), 4.90 (d, 0.3H) 4.39-4.13 (m,2H), 3.72 (s, 6H), 3.30 (s, 3H), 3.55-2.86 (m, 9H), 2.23-1.75 (m, 6H),1.16 (d, J=7.08 Hz, 3H), 1.10-1.45 (m, 6H), 0.95 (d, J=7.08 Hz, 3H). ¹³CNMR DMSO-d₆) δ=172.22, 170.42, 162.27, 158.06, 157.96, 145.04, 135.81,135.71, 135.34, 129.56, 127.75, 127.56, 126.57, 113.09, 85.09, 68.55,63.27, 54.97, 54.88, 50.57, 50.45, 35.74, 35.29, 30.74, 28.99, 28.96,28.89, 28.86, 28.71, 28.55, 28.47, 28.19, 27.29, 26.08, 25.16. MS.Molecular weight calculated for C₅₀H₇₃N₅O₆S₂, Cal. 904.27. Found 905.3(MH⁺).

Synthesis of the Amine 151.

Treatment of the azide 150 (1.86 g, 2.06 mmol) with triphenyl phosphine(0.54 g, 2.1 mmol) with THF (60 mL) and water (5 mL) at room temperaturefollowed by usual workup and column chromatography provided the pureamine 151 (1.45 g, 82%) as a foam. ¹H NMR (DMSO-d₆, 400 MHz) δ=7.93-7.89(m, 1H), 7.30-7.25 (m, 6H), 7.24-7.14 (m, 7H), 6.87-6.84 (m, 6H), 5.22(d, 0.7H), 4.90 (d, 0.3H) 4.39-4.13 (m, 2H), 3.72 (s, 6H), 3.30 (s, 3H),3.55-2.86 (m, 9H), 2.23-1.75 (m, 6H), 1.16 (d, J=7.08 Hz, 3H), 1.10-1.45(m, 6H), 0.95 (d, J=7.08 Hz, 3H). ¹³C NMR DMSO-d₆) δ=172.22, 170.42,162.27, 158.06, 157.96, 145.04, 135.81, 135.71, 135.34, 129.56, 127.75,127.56, 126.57, 113.09, 85.09, 68.55, 63.27, 54.97, 54.88, 50.57, 50.45,35.74, 35.29, 30.74, 28.99, 28.96, 28.89, 28.86, 28.71, 28.55, 28.47,28.19, 27.29, 26.08, 25.16. MS. Molecular weight calculated forC₅₀H₇₅N₃O₆S₂, Cal. 878.28. Found 879.26 (MH⁺).

Synthesis of compound 152

Using a similar procedure to that used for the synthesis of 146,coupling of the amine 151 (1.45 g, 1.65 mmol) with the acid 144 (1.02 g,1.65 mmol) provided the coupled product 152 (2 g, 81%) as a foam. ¹H NMR(DMSO-d₆, 400 MHz) δ=11.65 (bs, 1H), 8.90-8.85 (m, 1H), 7.93 (d, J=6 Hz,2H), 7.85 (m, H), 7.78 (d, J=76 Hz, 2H), 7.40-7.19 (m, 7H), 6.90-6.87(m, 6H), 5.21 (s, 2H), 4.80-4.56 (m, 1H), 4.42-4.32 (m, 1H), 4.26-4.16(m, 1H), 3.96-3.79 (m, 1H), 3.72 (s, 3H), 3.30-2.60 (m, 7H), 2.23-1.65(m, 6H), 1.36-1.15 (m, 24H), 1.12 (d, J=7 Hz, 6H). ¹³C NMR DMSO-d₆)δ=180.44, 172.32, 170.98, 170.80, 170.58, 165.51, 162.27, 159.10,158.21, 158.11, 155.53, 154.80, 149.82, 147.75, 145.60, 145.48, 141.75,136.45, 136.38, 136.33, 136.20, 134.21, 130.51, 129.68, 128.67, 128.45,127.89, 127.66, 126.70, 114.63, 113.27, 104.26, 85.94, 71.80, 61.70,57.32, 56.92, 5502, 53.99, 52.57, 52.46, 51.87, 50.45, 45.67, 38.63,38.44, 38.21, 35.74, 35.29, 30.74, 28.99, 28.96, 28.89, 28.86, 28.71,28.55, 28.47, 28.19, 27.29, 26.08, 25.16, 18.79, 11.16. MS. Molecularweight calculated for C₇₆H₉₉F₃N₁₀O₁₃S₂, Cal. 1481.78. Found 1481.0(M−H⁻).

The hydroxyl compound 152 on treatment with succinic anhydride followedby treatment with polystyrene linked resin provided the folate coupledsolid support 153 in 84 μM/g loading.

Synthesis of Compound 157

Using a similar procedure to that described above, a folate conjugatedsolid support with a cleavable disulfide linkage was synthesized asfollows. Treatment of the protected folic acid 144 with the amine 155provided the coupled precursor 156 as white foam.

Synthesis of Compound 156.

Using a similar procedure to that used for the synthesis of 146,coupling of the amine 155 (0.84 g, 1.45 mmol) with the acid 144 (0.875g, 1.41 mmol) provided the coupled product 156 (1.4 g, 83%) as a foam.¹H NMR (DMSO-d₆, 400 MHz) δ=8.89-8.85 (m, 2H), 8.08-7.62 (m, 5H),7.40-7.12 (m, 7H), 6.90-6.87 (m, 6H), 5.20 (s, 2H), 4.60-4.36 (m, 2H),4.20-4.12 (m, 1H), 3.72 (s, 6H), 3.30-2.60 (m, 7H), 2.23-1.65 (m, 6H),1.10 (d, J=7 Hz, 6H). ¹³C NMR DMSO-d₆) δ=180.35, 172.28, 171.32, 170.18,165.57, 160.52, 158.08, 157.96, 154.92, 149.65, 147.27, 145.04, 144.72,141.73, 135.83 135.70, 135.48, 134.28, 130.48, 129.57, 128.69, 128.46,127.85, 127.75, 127.55, 126.56, 114.63, 113.19, 113.09, 109.30, 85.10,68.55, 63.22, 54.97, 53.99, 52.38, 51.87, 45.67, 37.92, 37.18, 37.05,36.21, 34.87, 32.50, 31.58, 26.19, 24.01, 18.80, 11.08. MS. Molecularweight calculated for C₅₈H₆₄F₃N₉O₁₂S₂, Cal. 1200.31. Found 1200.10(M−H⁻).

The hydroxyl compound 156 on treatment with succinic anhydride followedby treatment with polystyrene linked resin provided the folate coupledsolid support 157 in 74 μM/g loading.

Synthesis of Compounds 163 and 164

Using a similar procedure to that described above, compounds 163 and 164are prepared. Treatment of the protected folic acid 144 with the amine161 provided the coupled precursor 162. The precursor 162 is thenconverted into a phosphoramidite, 164 and solid-support 163.

Synthesis of Compound 167 and 168

Using a similar procedure to that described above, compounds 167 and 168are prepared. Treatment of the protected folic acid 144 with the amine165 provided the coupled precursor 166. The precursor 166 is thenconverted into a phosphoramidite, 168 and solid-support 167.

Synthesis of Compounds 177 and 178

Using a similar procedure to that described above, compounds 174 and 175are prepared. Treatment of the protected folic acid 144 with the amine172 provided the coupled precursor 173. The precursor 173 is thenconverted into a phosphoramidite, 175 and solid-support 174.

Synthesis of Compounds 178

Synthesis of4(6-{2-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-[6-oxo-hexadecylcarbamoyl)-2-{4[(2-isobutrylamino-4-oxo-3,4-dihydro-pteridin-6-ylmethyl)-(2,2,2-trifluoro-acetyl)-amino]-benzoylamino}-butyricacid methyl ester 177

The pteroic acid precursor 144 (4.3 g, 6.92 mmol) was dissolved inanhydrous DMF (80 mL), HBTU (2.62 g, 1 eq.) followed by DIEA (2.7 g, 3eq.) were added and stirred for 20 minutes. To this reaction mixture theamine 176 (4.65 g, 6.92 mmol) was added as a solution in DMF (20 mL).Reaction was monitored by TLC (5% MeOH/DCM, PMA stain). TLC of thereaction mixture showed completion of the reaction after 1 h. Thereaction mixture was slowly poured in ice with vigorous stirring. Themixture was extracted with ethyl acetate and the combined organic layerswere dried (Na₂SO₄) and concentrated to give the crude product. The thusobtained crude product was further purified by column chromatography(silica gel, 0-5% MeOH in DCM in the presence of 1% NEt₃) to obtain 177as a white foam (Yield=5.1 g, 58%). ¹H NMR (DMSO-d₆, 400 MHz) δ=11.95(bs, 1H), 8.91-8.89 (m, 2H), 7.92 (d, J=6 Hz, 2H), 7.65 (m, H), 7.68 (d,J=76 Hz, 2H), 7.40-7.38 (m, 2H), 7.36-7.19 (m, 7H), 6.90-6.87 (m, 6H),5.21 (s, 2H), 4.80-4.56 (m, 1H), 4.42-4.32 (m, 1H), 4.26-4.16 (m, 1H),3.96-3.79 (m, 1H), 3.72 (s, 3H), 3.30-2.60 (m, 7H), 2.23-1.65 (m, 6H),1.36-1.15 (m, 24H), 1.12 (d, J=7 Hz, 6H). ¹³C NMR DMSO-d₆) δ=180.76,172.32, 170.98, 170.80, 170.58, 165.51, 162.27, 159.10, 158.21, 158.11,155.53, 154.80, 149.82, 147.75, 145.60, 145.48, 141.75, 136.45, 136.38,136.33, 136.20, 134.21, 130.51, 129.68, 128.67, 128.45, 127.89, 127.66,126.70, 114.63, 113.27, 104.26, 85.94, 71.80, 61.70, 57.32, 56.92, 5502,53.99, 52.57, 52.46, 51.87, MS. Molecular weight calculated forC₃₀H₃₄F₃N₇O₈, Cal. 1276.44. Found 1276.0 (M−H⁻).

Preparation of Solid Support 178:

Hydroxy derivative 177 (5 g, 3.91 mmol) was dissolved in DCM (100 mL) tothat Succinic anhydride (0.782 g, 2 eq) and DMAP (1.43 g, 3 eq.) wereadded and stirred overnight. TLC showed completion of reaction. Thereaction mixture was diluted with DCM (100 mL), washed successively withcold dilute citric acid and water (2 times), dried over sodium sulfate.Solvents were removed and dried under high vacuum to get the crudesuccinate. The thus obtained crude succinate was purified by columnchromatography (silica gel, 0-10% MeOH in DCM in the presence of 1% NEt₃to isolate the pure succinate as a white foam (4.7 g, 93%). Thissuccinate was dissolved in acetonitrile (100 mL) and to it PPh₃ (0.96 g,1.1 eq.), DMAP (0.61 g, 1.5 eq.) were added after which a solution ofDTNP (3.1 g, 1.05 eq.) in ACN (10 mL) was added to the above solution.The mixture was slowly shaken for 3-4 minutes. Long chain alkylamine-polystyrene (30 g, 250 mol/g) was added to the mixture and gentlyshaken for 2 h. The solid support was filtered, successively washed withDCM, mixture of MeOH/DCM (1:9) and DCM until filtrate remained colorlessand dried. The dried support was transferred into another flask treatedwith Ac₂O in pyridine (25%) in the presence of TEA (1 mL) for 15 min.under gentle shaking. Finally the solid support was filtered, washedwith DCM, DCM:MeOH (9:1), followed by DCM and ether. The solid support116 was dried under vacuum overnight and the loading was measured asreported (30 g, loading 72 mol/g).

Example 9 Synthesis of Folate Conjugates with PEG Spacer

In order to introduce a hydrophilic PEG spacer the following route wasused. Treatment of the commercially available PEGazido acid 179 with thehydroxyprolinol derivative 180 provided the coupled azide 181 which ontreatment with triphenyl phosphine in the presence of water provided theamine 182. The coupling of the amine 182 with the pteroic acid 113 togive the coupled product 183. The hydroxyl compound 183 on treatmentwith succinic anhydride followed by treatment with polystyrene linkedresin provided the folate coupled solid support 184 in 88 μM/g loading.

Preparation of Compound 181

Using a similar procedure to that used for the synthesis of 112,coupling of the amine 180 (2.1 g, 5 mmol) with the azido acid 179 (3.2g, 5 mmol) provided the coupled azide 181 (3.26 g, 64%) as a foam. ¹HNMR (DMSO-d₆, 400 MHz) δ=7.94-7.90 (m, 2H), 7.30-7.25 (m, 6H), 7.24-7.14(m, 7H), 6.87-6.84 (m, 6H), 5.22 (d, 0.7H), 4.90 (d, 0.3H) 4.39-4.13 (m,2H), 3.72 (s, 6H), 3.56-3.54 (m, 46H), 2.23-1.75 (m, 2H), 1.71-1.66 (m,2H). MS. Molecular weight calculated for C₅₃H₈₀N₄O₁₇, Cal. 1044.55.Found 1045.6 (MH⁺).

Synthesis of the Amine 182.

Treatment of the azide 181 (3.26 g, 3.12 mmol) with triphenyl phosphine(0.78 g, 3.1 mmol) with THF (60 mL) and water (5 mL) at room temperaturefollowed by usual workup and column chromatography provided the pureamine 182 (3.45 g, 82%) as a foam. ¹H NMR (DMSO-d₆, 400 MHz) δ=7.93-7.89(m, 1H), 7.30-7.25 (m, 6H), 7.24-7.14 (m, 7H), 6.87-6.84 (m, 6H), 5.22(d, 0.7H), 4.90 (d, 0.3H) 4.39-4.13 (m, 2H), 3.72 (s, 6H), 3.30 (s, 3H),3.55-2.86 (m, 9H), 2.23-1.75 (m, 6H), 1.16 (d, J=7.08 Hz, 3H), 1.10-1.45(m, 6H), 0.95 (d, J=7.08 Hz, 3H). MS. Molecular weight calculated forC₅₃H₈₂N₂O₁₇, Cal. 1119.22. Found 1120.26 (MH⁺).

Synthesis of Compound 183.

Using a similar procedure to that used for the synthesis of 112,coupling of the amine 182 (2.04 g, 2 mmol) with the acid 113 (1.24 g, 2mmol) provided the coupled product 183 (1.46 g, 46%) as a foam. ¹H NMR(DMSO-d₆, 400 MHz) δ=8.85 (s, 1H), 7.93 (d, J=6 Hz, 2H), 7.85 (m, H),7.78 (d, J=76 Hz, 2H), 7.40-7.19 (m, 7H), 6.90-6.87 (m, 6H), 5.21 (s,2H), 4.80-4.56 (m, 1H), 4.42-4.32 (m, 1H), 4.26-4.16 (m, 1H), 3.96-3.79(m, 1H), 3.72 (s, 3H), 3.62-3.46 (m, 48H), 2.23-1.65 (m, 6H), 1.36-1.15(m, 24H), 1.12 (d, J=7 Hz, 6H). MS. Molecular weight calculated forC₇₆H₁₀₆F₃N₉O₂₄, Cal. 1622.73. Found 1622.0 (M−H⁻).

Example 10 Synthesis of a Folate Analogue Conjugate 199

In order to evaluate the targeting ability of thepyrrolo[2,3-d]pyrimidine-based folate analogue, the multitargetedantifolate, Alimta [(a) Bunn, P. A., Jr.; Smith, I. E., Guest EditorsSeminars Oncol. 2002, 29 (6), Suppl. 18, 1-75. (b) Bertino, J.; Allegra,C.; Calvert, H., Guest Editors Seminars Oncol. 1999, 26 (2), Suppl. 6,1-111. (c) Hanauske, A.-R.; Chen, V.; Paoletti, P.; Niyikiza, C. TheOncologist 2001, 6, 363-373. (d) Taylor, E. C.; Kuhnt, D.; Shih, C.;Rinzel S. M.; Grindey, G. B.; Barredo, J.; Jannatipour, M.; Moran, R. G.J. Med. Chem. 1992, 35, 4450-4454.] the following approach wasundertaken.

The necessary building block for the conjugation to thepyrrolo[2,3-d]pyrimidine was synthesized as follows.

Synthesis of Compound 187

Caproic Acid (100 g, 0.7623 moles) was dissolved in 250 ml NaOH (2N) andcooled in a round bottom flask (2 L). Benzyl Chloroformate (273 mL,0.8004 moles) and 250 ml NaOH (2N) were added simultaneously at 0° C.with stirring. The progress of the reaction was monitored by TLC (about2 hr). After completion of the reaction, the mixture was acidified withdilute HCl (10%) and extracted with EtOAc (750 mL×3). The organic layerwas separated, washed well with water and dried over Na₂SO₄. Removal ofthe solvent under vacuum afforded crude 2 which was purified by columnchromatography (ethyl acetate:hexane). Yield: 80 g (40%). ¹H NMR (DMSO,400 MHz)=7.33 (s, 5H), 5.09 (s, 2H), 4.88 (br, 1H), 3.18 (m, 2H), 2.23(m, 2H), 1.62 (m, 2H), 1.50 (m, 2H), 1.34 (m, 2H).

Synthesis of Compound 188

Compound 187 (57.59 g, 0.2173 moles) was dissolved in 500 mL dry DCM andcooled to 0° C. in a round bottom flask (2 L). This was followed withthe addition of EDC.HCl (50 g, 0.2608 moles) and HOBt (33 g, 0.2173moles). After 15 minutes of stirring at 0° C., 186 (39.46 g, 0.2173moles) in 500 ml of dry DCM was added at 0° C. DIPEA was then added tillthe reaction mixture showed basic to pH. Reaction was stirred overnightat room temperature, quenched with water (500 mL) and extracted with DCM(500 mL×3). The organic layer was washed with sat. NH₄Cl and dried overanhydrous Na₂SO₄. Removal of solvent afforded the crude compound whichwas purified by column chromatography (4% MeOH/DCM) using 100-200 meshsilica gel. Yield 50 g (87%). ¹H NMR (DMSO, 400 MHz), δ=7.38 (m, 5H),7.25 (m, 1H), 5.18 (s, 1H), 4.99 (s, 2H), 4.32-4.25 (m, 2H), 3.67 (s,3H), 3.37 (m, 1H), 2.97 (m, 2H), 2.21 (m, 2H), 2.08 (m, 2H), 1.85 (m,1H), 1.46 (m, 2H), 1.39 (m, 2H), 1.25 (m, 2H). MS (MH)⁺: 393.35.

Synthesis of Compound 189

Compound 188 (135 g, 0.3440 moles) was dissolved in 800 mL THF in 3 LRBF. The reaction mixture was cooled to 0° C. and LiBH₄ (9.74 g, 0.4475moles) in 200 mL of THF was added in portion over a span of half hour.Reaction was stirred for additional half an hr at this temperature andthen at room temperature for 1.5 hr. The completion of the reaction wasmonitored by TLC. Reaction mixture was then cooled to 00° C. and dilutedwith 600 mL of water. 600 mL HCl (2N) was then added till pH was acidic.Excess of THF was removed under reduced pressure and residue wasextracted with EtOAc (700 mL×3). Organic layer was separated, washedwith brine and dried over Na₂SO₄. The solvent was stripped under reducedpressure to furnish 189 which was used directly for further reactions.Yield: 120 g (96%) ¹H NMR (DMSO, 400 MHz), δ=7.35 (m, 5H), 7.31 (m, 1H),4.98 (s, 2H), 4.27 (m, 1H), 4.02-3.95 (m, 1H), 3.47-3.22 (m, 6H)2.98-2.93 (m, 2H), 2.15 (m, 2H), 1.88 (m, 1H), 1.76 (m, 1H), 1.47 (m,2H), 1.40 (m, 2H), 1.17 (m, 2H). MS (MH)⁺: 365.2.

Synthesis of Compound 190

To a stirring solution of 189 (2 g, 0.005494 mole), triethylamine (1.5mL, 0.01153 moles) in DCM (10 mL), DMTr-Cl (2.047 gm, 0.006043 moles) in10 mL DCM was added dropwise and the reaction mixture stirred overnight(14 h). Reaction mixture was concentrated and the product was purifiedby column chromatography (EtOAc/hexane) using 100-200 silica gel.

(Note: Few drops of TEA were added while loading the silica gel onto thecolumn to reduce the acidic nature of silica gel). Yield 1.5 gm (41%).¹H NMR (DMSO, 400 MHz), δ=7.35-7.16 (m, 15H), 6.87 (m, 4H), 4.99 (s,2H), 4.38 (m, 1H), 4.13 (m, 1H), 3.72 (s, 6H), 3.57 (m, 1H), 3.15 (m,1H), 2.97 (m, 3H), 2.19 (t, 2H), 2.01 (m, 1H), 1.83 (m, 1H), 1.46-1.23(m, 7H). MS (MH)⁺: 667.50.

Synthesis of Compound 191

The Cbz protected amine 190 (38 g, 0.05705 moles) was dissolved in 250mL EtOAc/MeOH (3:1). After degassing, triethylamine (Catalytic) wasadded. This was followed by the addition of Pd/C (5.5 g, 15 Wt %, 10 Wt% Degussa type) and the reaction mixture was stirred for 3 hr underhydrogen atmosphere. After completion of reaction (monitored by TLC),the mixture was filtered through celite bed and washed with 300 mL (3:1EtOAc:MeOH). Filtrate was concentrated to give crude 191 which waspurified by column chromatography (5% MeOH/DCM) on 100-200 silica gel.(Note: Few drops of TEA were added while loading the silica gel onto thecolumn to reduce the acidic nature of silica gel). Yield: 28 g (93%)_HNMR (DMSO, 400 MHz), δ=7.32-7.17 (m, 9H), 6.85-6.89 (m, 4H), 4.38 (m,1H), 4.45 (m, 1H), 4.14 (m, 1H), 3.76 (s, 6H), 3.58 (m, 1H), 3.34-2.97(m, 7H), 2.22-2.18 (m, 2H), 2.02-1.98 (m, 2H), 1.83 (m, 1H), 1.48-1.14(m, 4H). MS (MH)⁺: 533.37.

Synthesis of Compound 193

To a stirring solution of 192 (1 g, 0.003386, moles) in 7 mL DCM,EDC.HCl (0.778 g, 0.004763 moles) was added at 0° C. under nitrogenatmosphere. After stirring for 5 minutes HOBt (0.518 g, 0.03385 moles)was added and the reaction mixture was stirred additionally for 15 mins.191 (1.8 g, 0.003386 moles) in 7 mL of DCM was then added to reactionmixture at the same temperature and stirring was continued. This wasfollowed by DIPEA till reaction mixture showed basic on pH paper (˜0.7mL). Stirring was continued overnight at room temperature. Aftercompletion of reaction, it was quenched with ice, extracted with DCM (15mL×3) and washed with 20 mL NH₄Cl solution. The organic layer wasseparated, dried over Na₂SO₄ and concentrated under reduced pressure.Purification by column chromatography (5% MeOH-DCM) using 100-200 silicagel furnished pure 193 in respectable yield. Yield: 2.3 g (85%). ¹H NMR(DMSO, 400 MHz), δ=7.78 (m, 2H), 7.36-7.16 (m, 14H), 6.85 (m, 4H), 4.99(m, 2H), 4.37 (m, 1H), 4.08 (m, 1H), 3.72 (s, 6H), 3.62-3.55 (m, 4H),3.14 (m, 1H), 2.98 (m, 3H), 2.50 (m, 2H) 2.20-1.92 (m, 8H), 1.45-1.23(m, 7H). MS (MH)⁺: 810.49.

Synthesis of Compound 194

The Cbz protected amine 193 (63 mg, 0.077 mmoles) was dissolved in 4 mL,EtOAc:MeOH (3:1). After degassing, triethylamine (catalytic) was added.This was followed by the addition of Pd/C (9.4 mg, 15 Wt %, 10 Wt %Degussa type) and the reaction was stirred for 3 hr under hydrogenatmosphere. The progress of the reaction was monitored by TLC. Aftercompletion (5 hrs) the reaction was filtered through celite bed andwashed with 15 mL (3:1 EtOAc:MeOH). The filtrate was concentrated toafford the crude product which was column purified (5% MeOH-DCM) on100-200 silica gel. Yield: 40 mg (78%) ¹H NMR (DMSO, 400 MHz), δ=7.77(m, 1H), 7.30 (m, 4H), 7.21-7.16 (m, 5H), 6.88 (m, 4H), 4.38 (m, 1H),4.25 (m, 1H), 3.72 (s, 6H), 3.60 (m, 4H), 3.33-3.16 (m, 2H), 3.15 (m,1H), 2.98 (m, 3H), 2.2 (t, 2H), 2.12 (t, 2H), 2.0 (m, 2H) 1.83 (m, 2H),1.6 (m, 1H), 1.46 (m, 2H), 1.37 (m, 2H), 1.25 (m, 4H). MS (MH)⁺: 676.41.

Synthesis of Compound 196

To suspension of Compound 195 (0.2 g, 0.000671 moles) in anhydrouspyridine (5 mL) was added DMAP (0.13 g, 0.0010 moles), followed byisobuytric anhydride (0.6 mL, 0.0040 moles) at room temperature. Theresulting mixture was then refluxed for 4 hr.

After completion of reaction (by TLC), the mixture was poured ontoice-HCl/hexane and stirred well. The resulting solid was filtered,washed with hexane and used directly for further reactions. Yield (0.1g, 34%). ¹H NMR (DMSO, 400 MHz): δ=12.08 (s, 1H), 11.48 (s, 1H), 7.85(d, 2H), 7.35 (d, 2H), 7.22 (s, 1H), 4.33 (m, 1H), 2.98 (m, 2H), 2.96(m, 2H), 2.81 (m, 1H), 1.19 (d, 6H), 1.14 (d, 6H). ¹³C NMR (DMSO):179.85, 175.21, 167.00, 156.64, 147.81, 147.34, 146.73, 129.08, 128.32,128.14, 121.27, 116.00, 106.07, 35.02, 34.51, 33.37, 26.90, 18.72,18.64. MS (MH): 439.40.

Synthesis of Compound 197

To a stirring solution of 196 (0.4 g, 0.00091 moles) in 5 ml MeOH, DIPEA(0.036 mL, 0.00278 moles), was added at room temperature. (Note: Afteraddition of DIPEA reaction mixture becomes clear). After 10 minutes DMAP(catalytic) was added to the mixture. The completion of reaction wasmonitored by TLC (& LCMS). MeOH was then concentrated and the residuewas diluted with water (5 mL). Acidification with dilute HCl wasfollowed by extraction with ethyl acetate. Organic layer was separated,dried over Na₂SO₄ and concentrated. The crude mixture was pure enoughand was used directly for further reactions. Yield: 260 mg (54%). ¹H NMR(DMSO, 400 MHz), δ=12.77 (bs, 1H), 11.68 (s, 1H), 11.34 (s, 2H), 7.84(d, 2H), 7.32 (d, 2H), 6.64 (s, 1H), 3.01 (m, 2H), 2.94 (m, 2H), 2.74(m, 1H), 1.09 (d, 6H). MS (MH)⁺: 369.10.

Synthesis of Compound 198

To a solution of 197 (4 g, 0.01086 moles) in 15 mL dry DMF, HBTU (4.11g, 0.01086 moles) and DIPEA (3.87 mL, 0.02173 moles) were added at roomtemperature under stirring. After half an hour, 194 (7.33 g, 0.01086moles) in dry DMF (10 mL) was added to the solution and stirring wascontinued. The progress of the reaction was monitored by TLC (4 hr).After completion, the reaction mixture was poured into crushed ice andwas extracted with EtOAc (100 mL×3). Organic layer was separated anddried over Na₂SO₄. Removal of solvent under reduced pressured afforded198, which was purified by column chromatography (3% MeOH/DCM). Yield:3.6 g (33%). ¹H NMR (DMSO, 400 MHz), δ=11.68 (s, 1H), 11.34 (bs, 2H),8.73 (bs, 1H), 7.83-7.77 (m, 3H), 7.29-7.16 (m, 11H), 6.87 (m, 4H), 6.63(s, 1H), 4.99 (m, 1H), 4.37 (m, 2H), 4.12 (bs, 1H), 3.71 (bs, 6H), 3.62(broad, 3H), 3.57 (m, 1H), 3.50 (m, 2H), 3.15 (m, 3H), 2.99 (m, 8H),2.74 (m, 2H), 2.18 (m, 4H), 1.44-1.22 (m, 6H), 1.11 (d, 6H) 1.09 (m,2H), 1.02 (m, 1H). MS (MH)⁺: 1026.60

The hydroxyl compound 198 on treatment with succinic anhydride followedby treatment with polystyrene linked resin provided the folate coupledsolid support 199 in 72 μM/g loading.

Example 11 Folate Building Blocks for Click-Chemistry

In order to synthesize azido functional group containing folateconjugates the following strategy was used. The azido amine tether 204was synthesized starting from the commercially available diamine 201 asshown below.

Synthesis of Amine 202

To a solution of the diamine (22 g, 0.1 mol) in dichloromethane (300mL), triethylamine (15 mL) was added and the mixture was cooled in icebath. To this cold solution a solution of (Boc)₂O in CH₂Cl₂ (100 mL) wasadded dropwise and the mixture was stirred overnight. The reactionmixture was washed with satd. NaHCO₃ (200 mL), water (300 mL), brine(300 mL) and dried (Na₂SO₄). Concentration of this organic layerfollowed by column purification provided the pure mono Boc amine 202 in55% yield. MS: MW Calc. for C₁₅H₃₂N₂O₅: 320.42. Found 321.41 (MH⁺).

Synthesis of Azide 203:

The triflic azide stock solution was prepared as reported in TetrahedronLetters 47 (2006) 2382-2385. The amine (0.96 g, 3 mmol), sodiumbicarbonate (0.85 mg, 10 mmol) and copper (II) sulfate pentahydrate (22mg, 0.1 mmol) were dissolved in water (3 mL). Triflic azide stocksolution (5 mL) was added, followed by the addition of methanol (20 mL)to yield a homogeneous system. The blue mixture was stirred for 30 minafter which the TLC and MS showed the complete disappearance of startingamine. The reaction mixture was concentrated in a rotary evaporator andthe residue was purified by chromatography on silica gel (eluent:dichloromethane-methanol) to obtain the pure azide 203 (1 g, 96%) as anoil. MS: MW Calc. for C₁₅H₃₀N₄O₅: 346.42. Found 347.41 (MH⁺). ¹HNMR(CDCl₃, 400 MHz) δ=4.68 (bs, 1H), 3.40-3.30 (m, 12H), 3.16 (t, J=6.4 Hz,2H), 3.00-2.95 (m, 2H), 1.68-1.54 (m, 4H), 1.04 (s, 9H).

Synthesis of 204:

The azide 203 (1 g, 2.88 mmol) was dissolved in ethanol (10 mL) and tothis a 2M solution of HCl in ether was added and the mixture was stirredat room temperature overnight. The MS showed the absence of startingmaterial. The reaction mixture was concentrated and the thus obtainedoil was used as such for the next reaction without further purification.MS: MW Calc. for C₁₀H₂₃ClN₄O₃: 246.17. Found 247.17 (MH⁺). ¹HNMR(DMSO-d₆ 400 MHz) δ=8.96 (bs, 1H), 7.92 (bs, 2H), 3.52-3.40 (m, 12H),3.37 (t, J=6.8 Hz, 2H), 2.85-2.77 (m, 2H), 1.81-1.70 (m, 4H).

Synthesis of 205:

Coupling of the amine 204 (0.6 g) with the acid 144 (1.2 g) using asimilar procedure to that used for the synthesis of 146 provided thecoupled azide 205 (1.68 g, 93%) as a light yellow foam. ¹H NMR (DMSO-d₆,400 MHz) δ=12.34 (s, 1H), 11.95 (s, 1H), 8.89 (s, 2H), 7.92 (d, J=8.4Hz, 2H), 7.81 (m, 1H), 7.70 (d, J=8.4 Hz, 2H), 5.22 (s, 2H), 4.40-4.34(m, 1H), 3.62 (s, 3H), 3.50-3.31 (m, 15H), 3.09-3.00 (m, 2H), 2.80-2.72(m, 1H), 2.20 (t, J=7.4 Hz, 2H) 2.10-1.89 (m, 2H), 1.76-1.54 (m, 4H),1.12 (d, J=6.8 Hz, 6H). MS. Molecular weight calculated forC₃₆H₄₆F₃N₁₁O₁₀, Cal. 849.81. Found 850.2 (MH⁺).

Synthesis of 206:

The azide 205 (1 g) was dissolved in THF (20 mL) and to it an aqueoussolution of lithium hydroxide (100 mg in 2 mL of water) was added andthe solution was stirred at room temperature for 4 h after which the MSshowed the complete disappearance of SM. The reaction mixture wasacidified to pH 5 using acetic acid and the RM was diluted with ethylacetate (100 mL). The precipitated product was filtered off and washedwith water and ethyl acetate and dried under vacuo at 40° C. overnightto get the pure azide 206 (0.455 g 55%) as an orange solid. ¹H NMR(DMSO-d₆, 400 MHz) δ=8.59 (s, 1H), 7.85 (bs, 1H), 7.72 (bs, 1H), 7.56(d, J=8.4 Hz, 2H), 6.88 (bs, 1H), 6.65 (d, J=8.4 Hz, 2H), 4.45 (s, 2H),4.00-4.02 (m, 1H), 3.50-3.33 (m, 14H), 3.04-3.00 (m, 2H), 2.07-1.83 (m,4H), 1.76-1.54 (m, 4H). MS. Molecular weight calculated for C₂₉H₃₉N₁₁O₈,Cal. 669.69. Found 668.2 (M−H⁻).

In another embodiment, the alkyne containing folic acid is synthesizedas follows. In this case the protected pteroic acid 144 was coupled withthe protected lysine 207 to get the coupled product 208 which on Cbzdeprotection provided the amine 209. Coupling of the amine 209 with theacid 210 provided the coupled product 211 which after purification anddeprotection provided the product 212 as described below.

Synthesis of 208:

Using a similar procedure to that used for the synthesis of 205,coupling of the acid 144 with the lysine derivative 207 provided thecoupling product 208 as a white solid in 95% yield.

Synthesis of 209:

The compound 208 on hydrogenation with Pd/C provided the deprotectedamine 209 as a yellow solid.

Synthesis of 210:

Coupling of the amine 209 with the acid 210 using a procedure to thatused for the synthesis of 205 provided the couple product 210 in highyields.

Synthesis of 212:

The deprotection of the protecting groups is achieved using a similarprocedure as described for the synthesis of 206 to isolate the fullydeprotected alkyne 212.

The synthesis of the building block 213 is then carried out as shownbelow.

The building block 213 is then converted to solid-support 214 orphosphoramidite 215.

Example 12 Synthesis of Folate Conjugate 216

In order to conjugate folic acid to the 5′ end of the oligo, postsynthetically, the following route was developed. Treatment of folicacid 215 with DCC followed by N-hydroxysuccinimide provided theactivated ester 216 in 80% yield. In a typical procedure, folic acid (5g, 11.33 mmol) was dissolved in anhydrous DMSO (100 mL) and to thissolution was added, triethyamine (2.5 mL), DCC (4.7 g, 22.6 mmol) andN-hydroxysuccinimide (2.6 g, 22.6 mmol) and the solution was stirred atroom temperature in dark for 18 h. The reaction mixture was filtered andto the filtrate EtOAc (1 L) was added and the precipitated product wasfiltered, washed with ethyl acetate (500 mL), ether (200 mL) and driedunder vacuum to isolate the product as a yellow powder. The purity ofthe product was found to be 83% by HPLC. This product was used as suchfor the coupling steps without further purification.

In another embodiment a cleavable acetal linkage was used in order tofacilitate the release of siRNA from the targeting folate as follows.

The ketal 217 was synthesized using a reported procedure (Paramonov, S.E.; Bachelder, E. M.; Beaudette, T. T.; Standley, S. M.; Lee, C. C.;Dashe, J.; Frechet, Jean M. J. Fully Acid-Degradable BiocompatiblePolyacetal Microparticles for Drug Delivery. Bioconjugate Chemistry(2008), 19 (4), 911-919). The transient protection of the ketal wascarried out in two steps in one pot first by treating the diamine withone equivalent of ethyltrifluoroacetate followed by one equivalent ofCbz-OSu to provide the di protected derivative 218 in 80% yield aftercolumn purification. The protected amine 218 on treatment with aqueousLiOH provided the amine 219 in quantitative yield. Coupling of thisamine 219 (0.5 g) with the protected folic acid 144 (1 g) provided thecoupled product 220 (1.1 g) which on hydrogenation provided the amine221 in quantitative yield.

Coupling of amine 221 was carried out with the maleimidopropionic acid222 to give the coupled product 223 in good yields. The finaldeprotection of all the protecting group in 223 is carried out usingice-cold aqueous LiOH in THF to afford the precursor 224 as an orangesolid.

Example 13 Synthesis of Conjugated siRNAs

Synthesis of Folate Conjugated siRNA:

The 3′-folate and C6-S—S—C6-folate siRNA conjugates with and withoutfluorophore label and its controls and corresponding antisense strandsused in this study are shown in Tables 4-7. These were individuallysynthesized using commercially available5′-O-(4,4′-dimethoxytrityl)-2′-O-t-butyldimethylsilyl-3′-O-(2-cyanoethyl-N,N-diisopropyl)RNA phosphoramidite monomers of 6-N-benzoyladenosine (A^(Bz)),4-N-acetylcytidine (C^(Ac)), 2-N-isobutyrylguanosine (G^(iBU)), anduridine (U), according to standard solid phase oligonucleotide synthesisprotocols as previously described (1). The folate conjugated strandswere synthesized using folate containing solid support. (C, FIG. 2). Theintroduction of cholesterol unit in the sequence was performed from ahydroxyprolinol-cholesterol phosphoramidite. Cholesterol was tethered totrans-4-hydroxyprolinol via a 6-aminohexanoate linkage to obtain ahydroxyprolinol-cholesterol moiety. 5′-end Cy-3 and Cy-5.5 (fluorophore)labeled siRNAs were synthesized from the corresponding Quasar-570 (Cy-3)phosphoramidite phosphoramidite purchased from Biosearch Technologies).The disulfide linker was introduced to the siRNA using C6-S—S—C6phosphoramidite as purchased from Glen Res. VA. An extended 15 mincoupling of 0.1M solution of phosphoramidite in anhydrous CH₃CN in thepresence of 5-(ethylthio)-1H-tetrazole activator to a solid boundoligonucleotide. Oxidation of the internucleotide phosphite to thephosphate was carried out using tert-butylhydroperoxide/acetonitrile/water (10:87:3) with 10 min oxidation waittime afforded folate conjugated oligonucleotide.

Deprotection-I

After completeness of oligonucleotide synthesis, a solid support wastreated with 1 M aq. piperidine for 24 h at room temperature. The solidsupport was washed with another portion of deprotecting reagent andcombined solutions were evaporated under reduced pressure. Added amixture of ethanolic ammonia [ammonia (28-30%): ethanol (3:1) 1.0 mL]for 8 h at 55° C. (2). The vial was cooled briefly on ice and then theethanolic ammonia mixture was transferred to a new microfuge tube. TheCPG was washed with portions of deionized water (2×0.1 mL). The combinedfiltrate was then put in dry ice for 10 min dried in a speed vac.

Deprotection-II (Removal of 2′-O-TBDMS Group)

The white residue obtained was resuspended in a mixture oftriethylamine, triethylamine trihydrofluoride (TEA.3HF ca, 24% HF) andDimethylsulfoxide (DMSO) (1:2:6)

(3.0 mL) and heated at 65° C. for 2 h to remove thetert-butyldimethylsilyl (TBDMS) groups at the 2′-position.

Analysis of Folate Conjugated siRNA:

The Folate conjugated sequences were analyzed by high-performance liquidchromatography (HPLC) on Phenomenox C6 phenyl column. The buffer A was50 mM TEAA, 10% Acetonitrile and Buffer B was 50 mM Sodium Acetate, 70%Acetonitrile at 65° C. in gradient of 25-80% B in 35 min

HPLC Purification:

The Folate conjugated, Cy-3, Cy 5.5 and Cy-3-Folate hybrid sequenceswere purified by high-performance liquid chromatography (HPLC) on anin-house packed RPC-Source15 reverse-phase column. The buffers were 20mM NaOAc in 10% CH₃CN (buffer A) and 20 mM NaOAc in 70% CH₃CN (bufferB). Fractions containing full-length oligonucleotides were pooled,desalted and lyophilized. Analytical HPLC, CGE and ES LC-MS establishedthe integrity of the compounds. The unconjugated oligonucleotides werepurified by anion-exchange HPLC on a TSK gel column packed in house. Thebuffers were 20 mM sodium phosphate (pH 8.5) in 10% CH₃CN (buffer A) and20 mM sodium phosphate (pH 8.5) in 10% CH₃CN, 1M NaBr (buffer B).Fractions containing full-length oligonucleotides were pooled, desalted,and lyophilized. For duplex generation, equimolar amounts of sense andantisense strand were heated in 1×PBS at 95° C. for 5 min and slowlycooled to room temperature.

Duplex Preparation

For the preparation of duplexes, equimolar amounts of sense andantisense strand were heated in 1×PBS at 95° C. for 5 min and slowlycooled to room temperature. Integrity of the duplex was confirmed byHPLC analysis.

Synthesized oligonucleotides and duplexes are shown in Table 4-7.

TABLE 4 Oligonucleotides synthesized and evaluated SEQ Duplex ID TargetID No S/AS Sequence 5′-3′ Luc AD-  30 A-30678 5′CcAcAuGAAGcAGcACGACuUQ51sL37 3618  31 A-4381 5′AAGUCGUGCUGCUUCAUGUGgsusC Luc AD-  32 A-30677 5′AccGAAAGGucuuAccGGAdTdTQ51sL37 3614  33 A-30674 5′UCCGGuAAGACCUUUCGGUdTdTsL10 Luc AD-  34 A-30659 5′Q38cuuacgcugaguacuucgadTdTL37 3514  35 A-30660 5′ucgaaguacucagcguaagdTdTL8 3′ Luc AD-  36 A-30659 5′Q38cuuacgcugaguacuucgadTdTL37 3515  37 A-30661 5′ucgaaguacucagcguaagdTdT-L10 GFP AD-  38 A-30678 5′CcAcAuGAAGcAGcACGACuUQ51sL37 3615  39 A-3389 5′AAGUCGUGCUGCUUCAUGUGgusCs-L10 GFP AD-  40 A-30676 5′cuGGcuGAAuuucAGAGcAdTdTQ51sL37 3616  41 A-22826 5′UGCUCUGAAAUUcAGCcAGdTsdT Luc AD-  42 A-30677 5′AccGAAAGGucuuAccGGAdTdTQ51sL37 3617  43 A-24775 5′UCCGGuAAGACCUUUCGGUdTsdT CD45 AD-  44 A-30676 5′cuGGcuGAAuuucAGAGcAdTdTQ51sL37 3613  45 A-30673 5′UGCUCUGAAAUUcAGCcAGdTdTs L10 CD45 AD-  46 A-228255′-cuGGcuGAAuuucAGAGcAdTsdT 3637  47 A-30687 5′Q39UGCUCUGAAAUUcAGCcAGdTsdT CD45 AD-  48 A-228255′-cuGGcuGAAuuucAGAGcAdTsdT 3638  49 A-30695 5′Q39UGCUCUGAAAUUcAGCcAGdTdTsL10 CD45 AD-  50 A-30676 5′cuGGcuGAAuuucAGAGcAdTdTQ51sL37 3639  51 A-30852 5′Q78UGCUCUGAAAUUcAGCcAGdTsdT CD45 AD-  52 A-30676cuGGcuGAAuuucAGAGcAdTdTQ51sL37 3633  53 A-30821 5′UGCUCUGAAAUUcAGCcAGdTsdTL10 CD45 AD-  54 A-228255′-cuGGcuGAAuuucAGAGcAdTsdT 3634  55 A-30821 5′UGCUCUGAAAUUcAGCcAGdTsdTL10 CD45 AD-  56 A-306765′cuGGcuGAAuuucAGAGcAdTdTQ51sL37 3635  57 A-30687 5′Q39UGCUCUGAAAUUcAGCcAGdTsdT CD45 AD-  58 A-30676 5′cuGGcuGAAuuucAGAGcAdTdTQ51sL37 3636  59 A-30695 5′Q39UGCUCUGAAAUUcAGCcAGdTdTsL10 CD45 AD-  60 A-30592 5′cuGGcuGAAuuucAGAGcAdTdTQ51L37 3599  61 A-22826 5′UGCUCUGAAAUUcAGCcAGdTsdT CD45 AD-  62 A-31653 5′cuGGcuGAAuuucAGAGcAdTsdTL37 18744  63 A-30687 5′Q39UGCUCUGAAAUUcAGCcAGdTsdT CD45 AD-  64 A-31632 5′cuGGcuGAAuuucAGAGcAUUZ32 18218  65 31633 and 5′ UGCUCUGAAAUUcAGCcAGUU 66 31634 5′Q67dAdAdCdCdGdTdGdGdTdCdAdTdGdCdT dCdC GFP AD-  67 A-316355′ CcAcAuGAAGcAGcACGACUUUUZ32 18219  68 31636 and 5′UGCUCUGAAAUUcAGCcAGUU  69 31634 5′ Q67dAdAdCdCdGdTdGdGdTdCdAdTdGdCdTdCdC CD45 AD-  70 A-31653 5′cuGGcuGAAuuucAGAGcAdTsdTL37 18367  71 A-310155′ugcucugaaauucagccagdTsdTL63 GFP AD-  72 A-33204 5′AcAuGAAGcAGcACGACuUdTdTQ11L37 18860  73 A-32594 5′AAGUCGUGCUGCUUCAUGUdTsdT GFP AD-  74 A-33169 5′AcAuGAAGcAGcACGACuUdTsdTL37 18866  75 A-32594 5′AAGUCGUGCUGCUUCAUGUdTsdT GFP AD-  76 A-33169 5′AcAuGAAGcAGcACGACuUdTsdTL37 18866  77 A-32594 5′AAGUCGUGCUGCUUCAUGUdTsdT GFP AD-  78 A-321415′CcAcAuGAAGcAGcACGACusUL102 18576  79 A-4381 5′AAGUCGUGCUGCUUCAUGUGgsusC GFP AD-  80 A-32142 5′CcAcAuGAAGcAGcACGACusUL103 18578  81 A-4381 5′ AAGUCGUGCUGCUUCAUGUGgsusCGFP AD-  82 A-32142 5′ CcAcAuGAAGcAGcACGACusUL103 18579  83 A-32145 5′AAGUCGUGCUGCUUCAUGUGgsusCL10 GFP AD-  84 A-32143 5′CcAcAuGAAGcAGcACGACusUL104 18580  85 A-4381 5′ AAGUCGUGCUGCUUCAUGUGgsusCGFP AD-  86 A-32143 5′ CcAcAuGAAGcAGcACGACusUL104 18581  87 A-32145 5′AAGUCGUGCUGCUUCAUGUGgsusCL10 GFP AD-  88 A-32593 5′AcAuGAAGcAGcACGACuUdTsdT 18747  89 A-32592 5′ AAGUCGUGCUGCUUCAUGUdTdTL48GFP AD-  90 A-33199 5′ AcAuGAAGcAGcACGACuUdTdTL10 18858  91 A-32594 5′AAGUCGUGCUGCUUCAUGUdTsdT GFP AD-  92 A-33205 5′Q11AcAuGAAGcAGcACGACuUdTsdT 18861  93 A-32594 5′AAGUCGUGCUGCUUCAUGUdTsdT GFP AD-  94 A-33199 5′AcAuGAAGcAGcACGACuUdTdTL10 18862  95 A-32592 5′AAGUCGUGCUGCUUCAUGUdTdTL48 GFP AD-  96 A-33169 5′AcAuGAAGcAGcACGACuUdTsdTL37 18867  97 A-32592 5′AAGUCGUGCUGCUUCAUGUdTdTL48 GFP AD-  98 A-33200 5′AcAuGAAGcAGcACGACuUdTdTL103 19084  99 A-32592 5′AAGUCGUGCUGCUUCAUGUdTdTL48 GFP AD- 100 A-33201 5′Q11AcAuGAAGcAGcACGACuUdTdTL103 19085 101 A-32592 5′AAGUCGUGCUGCUUCAUGUdTdTL48 GFP AD- 102 A-33203 5′AcAuGAAGcAGcACGACuUdTdTQ11L103 19086 103 A-32592 5′AAGUCGUGCUGCUUCAUGUdTdTL48 GFP AD- 104 A-33201 5′Q11AcAuGAAGcAGcACGACuUdTdTL103 19087 105 A-32594 5′AAGUCGUGCUGCUUCAUGUdTsdT GFP AD- 106 A-33203 5′AcAuGAAGcAGcACGACuUdTdTQ11L103 19088 107 A-32594 5′AAGUCGUGCUGCUUCAUGUdTsdT GFP AD- 108 A-33054 5′CcAcAuGAAGcAGcACGACusUL113 19185 109 A-4381 5′ AAGUCGUGCUGCUUCAUGUGgsusCGFP AD- 110 A-32141 5′ CcAcAuGAAGcAGcACGACusUL102 18577 111 A-32145 5′AAGUCGUGCUGCUUCAUGUGgsusCL10 GFP AD- 112 A-33202 5′Q11AcAuGAAGcAGcACGACuUdTsdT 18859 113 A-32594 5′AAGUCGUGCUGCUUCAUGUdTsdT GFP 114 A-34122 AcAuGAAGcAGcACGACuUdTsdTL10 115A-32594 AAGUCGUGCUGCUUCAUGUdTsdT GFP 116 A-34123AcAuGAAGcAGcACGACuUdTsdTL103 117 A-32594 AAGUCGUGCUGCUUCAUGUdTsdT GFP118 A-34124 Q11AcAuGAAGcAGcACGACuUdTsdTL103 119 A-32594AAGUCGUGCUGCUUCAUGUdTsdT GFP 120 A-34125 Q11AcAuGAAGcAGcACGACuUdTsdTL37121 A-32594 AAGUCGUGCUGCUUCAUGUdTsdT GFP 122 A-34126AcAuGAAGcAGcACGACuUdTsdTQl1L103 123 A-32594 AAGUCGUGCUGCUUCAUGUdTsdT GFP124 A-34127 AcAuGAAGcAGcACGACuUdTsdTQl1L37 125 A-32594AAGUCGUGCUGCUUCAUGUdTsdT Note: Lower case = 2′-OMe, S = PS linkage, L37:= N-(folic acidcarboxamidocaproyl)-4-hydroxyprolinol (Hyp-C6-folate),L63: = Oregon Green488-aminohexylcarboxamidocaproyl-prolinol-4-phosphate, Q8: =N-(aminocaproyl)prolinol-4-phosphate, L8: =N-(cholesterylcarboxamidocaproyl)-3-hydroxy-4-hydroxymethylpyrrolidine,Q38: = Quasar 570 phosphate (BNS-5063, Biosearch Tech), L10: =N-(cholesterylcarboxamidocaproyl)-4-hydroxyprolinol (Hyp-C6-Chol), Q39:= Quasar 705 phosphate (BNS-5067, Biosearch Tech), Q78: =Atto-610-aminohexylphosphate, L102: = N-(folatecarboxamidohexadecanoylcarboxamidoethyl-dithio-1,1-dimethylbutyryl)-4-hydroxyprolinol(Hyp-Me2S-S-C16-folate), L103: = N-(folatecarboxamidoethyl-dithio-butyryl)-4-hydroxyprolinol (Hyp-S-S-folate),L104: = N-(folate carboxamidohexadecanoyl)-4-hydroxyprolinol(Hyp-C16-folate), Q11: =N-(cholesterylcarboxamidocaproyl)prolinol-4-phosphate, Q51: =6-hydroxyhexyldithiohexylphosphate (Thiol-Modifier C6 S-S Glen Res.10-1936), L48: = N-(Alexa647-carboxamidocaproyl)-4-hydroxyprolinol(Hyp-C6-Alexa647), L113: = N-(folatecarboxamido-PEG12)-4-hydroxyprolinol (Hyp-PEG12-folate), Q67: =Folate-gamma-carboxamidohexylphosphate (Folate C6), Z32: =GGAGCAUGACCACGG (SEQ ID NO: 126).

TABLE 5 Oligonucleotides synthesized. SEQ ID Cal Found NoSequence (5′-3′) Mass Mass AL-3435 127 5′cuG AAG Acc uGA AGA cAA uTT-s-Folate 7529.02 7530.8 AL-3436 128 5′AUu GUC uUc AGG UCu UcA GTT-s-Folate 7385.76 7386.8 AL-3437 129 5′GAA CUG UGU GUG AGA GGU CCU-s-Folate 7499.7 7498.7 AL-3482 130 5′GAA CUG UGU GUG AGA GGU CCU chol s Folate 8203.7 8205.2 AL-3480 131 5′cuG AAG Acc uGA AGA cAA uTT-Chol-s-Folate 8232.3 8234.4 AL-3481 132 5′GAA CUG UGU GUG AGA GGU CCU-Chol-s-Folate 8203.7 8205.3 AL-3609 133 5′CUU ACG CUG AGU ACU UCG AdTdT-Chol-C18-Folate 3′ 8369.2 8371.70 AL-3610134 5′ Quasar CUU ACG CUG AGU ACU UCG AdTdT-Chol-C18- 8989.2 8990.15Folate3′ AL-3611 135 5′ CUU ACG CUG AGU ACU UCG AdTdT-C18-Chol-C18-8715.96 8715.30 Folate 3′ AL-3612 136 5′Quasar CUU ACG CUG AGU ACU UCG AdTdT-C18-Chol- 9333.96 9334.36C18-Folate3′ AL-3639 137 5′ CUU ACG CUG AGU ACU UCG ATT-C12S-S-Folate3′7650.05 7650.26 AL-3640 138 5′Quasar CUU ACG CUG AGU ACU UCG ATT-C12S-S-Folate 8270.26 8270.18 3′AL-3664 139 5′ CUU ACG CUG AGU ACU UCG ATT-C12S-S-C18-Folate 3′ 7994.357994.71 AL-3665 140 5′ Quasar CUU ACG CUG AGU ACU UCG ATT-C12S-S-C18-8614.67 8614.47 Folate3′ AL-3668 141 5′cuG AAG Acc uGA AGA cAA uTT C12S-S-Folate 3′ 7841.35 7841.37 AL-3669 1425′ Quasar cuG AAG Acc uGA AGA cAA uTT C12S-S- 8461.59 8461.47 Folate 3′AL-3670 143 5′ cuG AAG Acc uGA AGA cAA uTT C12S-S-C18-Folate3′ 8185.688186.05 AL-3671 144 5′ Quasar cuG AAG Acc uGA AGA cAA uTT C12S-S-C18-8805.99 8805.83 Folate 3′ Quasar = Cy3; C18 = Spacer C18 linker , C12S-S = disulfide linker, Chol = Cholesterol, lower case = 2′-o-Me, s =phosphorothioate linkage, Folate = Folate. See FIG. 1-8 for a graphicalrepresentation of sequences with the associated conjugates and structureof monomers used.

TABLE 6 Oligonucleotides synthesized. SEQ ID Cal Found NoSequence (5′-3′) Mass Mass Al-30658 145 5′Cy-3 cuuacgcugaguacuucgadTdT-Hyp-NH₂ 7786.5 7784.3 Al-30660 146 5′ucgaaguacucagcguaagdTdT-Hyp-NH₂ 3′ 7251.1 7250.6 Al-30659 147 5′Cy-3 cuuacgcugaguacuucgadTdT-Folate 3′ 8209.9 8207.8 Al-30661 148 5′ucgaaguacucagcguaagdTdT-cholesterol 76634.4 7663.6 Al-30676 149 5′cuGGcuGAAuuucAGAGcAdTdT-C6-S-S-C6-s-Folate 7856.4 7855.4 Al-30677 150 5′AccGAAAGGucuuAccGGAdTdT- C6-S-S-C6-s- 7864.4 7863.4 Folate Al-30678 1515′ CcAcAuGAAGcAGcACGACuU- C6-S-S-C6-s- 7822.3 7821.2 Folate Al-30673 1525′ UGCUCUGAAAUUcAGCcAGdTdTs-cholesterol 7379.0 7377.1 Al-30674 153 5′UCCGGuAAGACCUUUCGGUdTdTs-cholesterol 7357.9 7357.1 Al-3389 154 5′AAGUCGUGCUGCUUCAUGUGgusCs-cholesterol 8082.4 8081.1 Al-24775 155 5′UCCGGuAAGACCUUUCGGUdTsdT 6653.1 6652.3 Al-4381 156 5′AAGUCGUGCUGCUUCAUGUGgsusC 7277.5 7376.6 Al-30687 157 5′Cy-5.5 UGCUCUGAAAUUcAGCcAGTsT 7421.02 7420.4 Al-30695 158 5′Cy-5.5 UGCUCUGAAAUUcAGCcAGTTs- 8125.9 8124.6 cholesterol Hyp-NH2 =Hydroxy prolinol linker; Cy3 = Cy3 dye; Cy5.5 = Cy-5.5 dye; C6 S-S-C6 =disulfide linker; Chol = Cholesterol; lower case = 2′-O-Me; Folate =Folate; s = phosphorothioate linkage.

TABLE 7 Some more folate-conjugated duplexes. Al- SEQ Duplex ID Duplex #No Sequences Concentration AD-3513 159 5′Cy-3 cuuacgcugaguacuucgadTdT-Hyp-NH₂ 10 mg/ml 160 5′ucgaaguacucagcguaagdTdT-Hyp-NH₂ 3′ AD-3514 161 5′Cy-3 cuuacgcugaguacuucgadTdT-Folate 3′ 10 mg/ml 162 5′ucgaaguacucagcguaagdTdT-Hyp-NH₂ AD-3515 163 5′Cy-3 cuuacgcugaguacuucgadTdT-Folate 10 mg/ml 164 5′ucgaaguacucagcguaagdTdT-Hyp-Cholesterol Hyp-NH2 = Hydroxy prolinollinker; Cy3 = Cy3 dye; Chol = Cholesterol; lower case = 2′-O-Me; Folate= Folate;

Example 14 Binding Competition Assays

Folate conjugated siRNAs were incubated with KB cells in presence ofFTIC-folate (PBS, on Ice). % competition for folate receptor (FR) wasplotted as a measure of activity. FIG. 10 shows that different folateconjugates had different binding competition but sequence of theoligonucleotide made no difference on binding competition. The Kd werecalculated from the binding assays and are reported in table 8. The 3′sense folate with cleavable or non-cleavable linkers shows loweraffinity for FR than folic acid. Introduction of 3′ antisensecholesterol improves binding by ˜50 fold. 5′ folate attached to aDNA-oligo tether binds to FR with an affinity similar to that of folicacid. FR ligands have a slow off-rate—no significant competition isobserved if FITC-folate is added 1^(st).

TABLE 8 Folate receptor binding of folate conjugated siRNAs duplex #sense antisense KD (uM) AD-18218 5′ extended 5′ folate DNA oligo 0.3AD-18219 5′ extended 5′ folate DNA oligo 0.2 AD-185763′-Hyp-Me2S-S-C2-C16-folate unconjugated 12.5 AD-18578 3′-Hyp-S-S-folateunconjugated 34.8 AD-18579 3′-Hyp-S-S-folate 3′ chol 0.3 AD-185803′-Hyp-C16-folate unconjugated 20.7 AD-18581 3′-Hyp-C16-folate sense 3′chol 0.3 AD-3614 3′ C6-SS-C6-Hyp folate 3′ chol 0.2 AD-3617 3′C6-SS-C6-Hyp folate unconjugated 13.7

Example 15 Folate GFP Duplexes, In Vitro Silencing with TransfectionReagent

KB pEGFP clone 17 cells were used for all GFP silencing experiments. Forsilencing experiments with transfection reagent HiPerFect was used totransfect in the siRNAs tested and GFP expression was analyzed 72 hrslater. As seen in FIG. 11, presence of folate ligands (multiple linkerdesigns) at the 3′ of the sense strand does not significantly impactsilencing activity. 3′ antisense cholesterol decreased activity by 10×or greater depending on design, similar trends were observed for CD45duplexes. Addition of 3′ sense folate to 3′ or 5′ sense cholesterolduplex does not significantly alter silencing activity.

Example 16 Free Uptake Silencing Activity

Silencing by free uptake was evaluated in the KB-EGFP cells, a cell linestably expressing the EGFP gene. KB-EGFP cells were culturedcontinuously in folate free media with 10% FBS. For free uptakesilencing folate-siRNA conjugates were added to KB-EGFP cells in 24-wellplates in media with or without serum. For the “no serum” wells completemedia (with 10% FBS) was added 4 hrs later. Cells were cultured for 72hrs, removed from plates using Versene, washed and GFP expressionquantified by flow cytometry on a LSRII instrument. Data was analyzedusing the FlowJo software and median fluorescence intensity of the GFPsignal was plotted. As seen in Table 9, various tethers and linkerdesigns containing folate conjugates can be accommodated at the 3′-endof the sense strand without affecting RNAi activity. 25% silencing byfree uptake is observed for folate conjugated siRNA at 5 uMconcentration independent of serum. siRNAs comprising both cholesteroland folate show 40% silencing by free uptake in absence of serum at 5uM.

TABLE 9 Free uptake of folate conjugated siRNAs % free uptake silencingKD IC₅₀ (5 uM, no duplex # sense antisense (uM) (nM) serum) singeoverhang AD-5179 unconjugated unconjugated ND 0.09 0 AD-3618 3′C6-SS-C6-Hyp folate unconjugated ND 0.13 0 AD-3615 3′ C6-SS-C6-Hypfolate 3′ chol ND 0.69 0 AD-18576 3′-Hyp-Me2S-S-C2-C16-folateunconjugated 12.5 0.12 10 AD-18577 3′-Hyp-Me2S-S-C2-C16-folate 3′ cholND 4.09 ND AD-18578 3′-Hyp-S-S-folate unconjugated 34.8 0.09 0 AD-185793′-Hyp-S-S-folate 3′ chol 0.3 0.80 20 AD-18580 3′-Hyp-C16-folateunconjugated 20.7 0.14 0 AD-18581 3′-Hyp-C16-folate sense 3′ chol 0.32.04 0 double overhang AD-18527 unconjugated unconjugated ND 0.03 0AD-18526 unconjugated 3′ chol ND 2.08 ND AD-18858 3′ cholesterolunconjugated ND 0.07 80 AD-18861 5′ cholesterol unconjugated ND 0.06 40AD-18859 5′ cholesterol, 3′ folate unconjugated ND 0.07 40 AD-18860 3′cholesterol, 3′ folate unconjugated ND 0.04 40 AD-18866 3′ Hyp-C6 folateunconjugated ND 0.03 25 3-oligo duplex AD-18219 5′ extended 5′ folateDNA oligo 0.2 0.16 0

Example 17 Serum Stability of Folate Conjugated Oligonucleotides

siRNAs comprising folate conjugates with a cleavable linker wereincubated in mouse serum at 37° C. for 0, 0.5, 1, 2, 6, 16 and 24 hours.After incubation, amount of individual strands were quantified by IEXHPLC. Sense strand t_(1/2) decreased by 5-78× depending on the sequence.Table 10 summarizes the t_(1/2) of individual strands of tested siRNAs.For a 3′sense folate a gem-dimethyl linker duplex is more stable inserum then an unhindered disulfide linked duplex. The 3′ sense folateduplex with a C16 non-cleavable linker is as stable as the unmodifiedparent duplex. The 3′ sense folate duplexes with a Glen researchdisulfide linker show the lowest serum stability, however thesemolecules also lack the PS group in the overhang.

TABLE 10 Serum stability of folate conjugated duplexes in mouse serum(table discloses SEQ ID NOS 165-178, respectively, in order of appearance)Single Strand Half-life Duplex Strand Type 5-3 Full Strand (h) AD-321522825 s 5′-cuGGcuGAAuuucAGAGcAdTsdT 6.58 22826 as 5′UGCUCUGAAAUUcAGCcAGdTsdT 1.83 AD-3616 30676 s5′-cuGGcuGAAuuucAGAGcAdTdT-C6-S-S-C6-folate 1.22 22826 asUGCUCUGAAAUUcAGCcAGdTsdT 2.90 AD-5179 4545-b1 s CcAcAuGAAGcAGcACGACusU15.00 4381-b11 as AAGUCGUGCUGCUUCAUGUGgsusC 11.50 AD-3618 30678 s 5′CcAcAuGAAGcAGcACGACuU-C6-S-S-C6-Folate 0.19 4381 asAAGUCGUGCUGCUUCAUGUGgsusC 1.59 AD-18576 32141 sCcAcAuGAAGcAGcACGACusU-Hyp-Me2S-S-C16-folate 15.36 4381 asAAGUCGUGCUGCUUCAUGUGgsusC 8.22 AD-18578 32142 sCcAcAuGAAGcAGcACGACusU-Hyp-S-S-folate 6.39 4381 asAAGUCGUGCUGCUUCAUGUGgsusC 4.69 AD-18580 32143 sCcAcAuGAAGcAGcACGACusU-Hyp-C16-folate 15.72 4381 asAAGUCGUGCUGCUUCAUGUGgsusC 10.44

Example 18 In Vivo Folate Conjugate Distribution in KB Xenograft TumorModel

KB cells were injected subcutaneously on both flanks of Nu/Nu mice(3×10⁶ cells per site). Tumors were allowed to develop for 2 weeks. Micewere injected with 60 nmol fluorophore and folate conjugated siRNAs.Mice were saced at the desired time point and tumors and kidneysharvested for imaging on the Maestro imaging system and downstreamprocessing. For lysate assays tissues were frozen and ground to powderusing a mortar and pestle. Tissue powders (20 mg) were lysed using theMammalian Cell Lysis Kit (Sigma). Protein concentration was quantifiedusing the BCA assay, Oregon Green fluorescence in lysates was quantifiedusing the Victor plate reader. For tumor sections, animals were perfusedwith 4% PFA upon sacrifice, tumors were harvested and cryoprotected in30% sucrose. Tumors were sectioned by cryostat. Sections were post-fixedwith PFA and stained for mouse CD11b or F_(4/80) macrophage markers.Sections were imaged on the Axiovision fluorescence microscope andfluorescence normalized to protein concentration. Conjugated siRNAsAD-18367 (3′ non-cleavable folate sense and 3′ OregonGreen antisense)and AD-3716 (3′ OregonGreen antisense) were used. Figure x shows theaccumulation of folate conjugated siRNA (AD-18367) in the tumor andkidney at 4 hours. FIG. 12 show tissue uptake of folate conjugated siRNAat 4 hour and 18 hours after injection. As can be seen in FIG. 13,folate conjugated siRNA was present in significant amount in the tumorafter 18 hours. FIG. 14 shows the accumulation of Cy3 labeled folateconjugated siRNAs in tumors. Accumulation was only seen when the siRNAhad a folate conjugate, thus confirming targeting by folate conjugates.

Sections from a tumor treated with folate conjugated Cy3 labeled siRNA(AD-3514) were strained with an antibody to mouse F4/80 or an isotypecontrol. Results are shown in FIG. 15.

Example 19 KB EGFP Tumors in Nude Mice

Nu/Nu mice were SC implemented with KB or KB EGFP cells. Tumors wereimaged on the Maestro system. Tumor lysates were prepared to measure GFPlevels. FIG. 16 shows results of tumor establishment and GFP levels inthe tumors.

We claim:
 1. An iRNA agent comprising at least one monomer having thestructure shown in formula (I′)

wherein: A and B are each independently for each occurrence O, N(R^(N))or S; X is H, a protecting group, a phosphate group, a phosphodiestergroup, an activated phosphate group, an activated phosphite group, aphosphoramidite, a solid support, —P(Z′)(Z″)O-nucleoside,—P(Z′)(Z″)O-oligonucleotide, a lipid, a PEG, a steroid, a polymer,—P(Z′)(Z″)O-L⁶-Q′-L⁷-P(Z′″)(Z″″)O-oligonucleotide, a nucleotide, or anoligonucleotide; Y is H, a protecting group, a phosphate group, aphosphodiester group, an activated phosphate group, an activatedphosphite group, a phosphoramidite, a solid support,—P(Z′)(Z″)O-nucleoside, —P(Z′)(Z″)O-oligonucleotide, a lipid, a PEG, asteroid, a lipophile, a polymer,—P(Z′)(Z″)O-L⁶-Q′-L⁷-OP(Z′″)(Z″″)O-oligonucleotide, a nucleotide, or anoligonucleotide; R is folate, a folate analog a folate mimic or a folatereceptor binding ligand; L⁶ and L⁷ are each independently for eachoccurrence —(CH₂)_(n)—, —C(R′)(R″)(CH₂)_(n)—, —(CH₂)_(n)C(R′)(R″)—,—(CH₂CH₂O)_(m)CH₂CH₂—, or —(CH₂CH₂O)_(m)CH₂CH₂NH—; Q′ is NH, O, S, CH₂,C(O)O, C(O)NH, —NH—CH(R^(a))—C(O)—, —C(O)—CH(R^(a))—NH—, CO,

R^(a) is H or amino acid side chain; R′ and R″ are each independently H,CH₃, OH, SH, NH₂, NH(Alkyl) or N(diAlkyl); Z′, Z″, Z′″ and Z″″ areindependently O or S; n represent independently for each occurrence1-20; and m represent independently for each occurrence 0-50.
 2. An iRNAagent comprising at least one monomer having the structure shown informula (I)

wherein: X is H, a hydroxyl protecting group, a phosphate group, aphosphodiester group, an activated phosphate group, an activatedphosphite group, a phosphoramidite, a solid support,—P(Z′)(Z″)O-nucleoside, —P(Z′)(Z″)O-oligonucleotide, a lipid, a PEG, asteroid, a polymer, —P(Z′)(Z″)O-L⁶-Q′-L⁷-OP(Z′″)(Z″″)O-oligonucleotide,a nucleotide, or an oligonucleotide; Y is H, a hydroxyl protectinggroup, a phosphate group, a phosphodiester group, an activated phosphategroup, an activated phosphite group, a phosphoramidite, a solid support,—P(Z′)(Z″)O-nucleoside, —P(Z′)(Z″)O-oligonucleotide, a lipid, a PEG, asteroid, a lipophile, a polymer,—P(Z′)(Z″)O-L⁶-Q′-L⁷-OP(Z′″)(Z″″)O-oligonucleotide, a nucleotide, or anoligonucleotide; Q is a tether; R is folate, a folate analog a folatemimic or a folate receptor binding ligand; L⁶ and L⁷ are eachindependently for each occurrence —(CH₂)_(n)—, —C(R′)(R″)(CH₂)_(n)—,—(CH₂)_(n)C(R′)(R″)—, —(CH₂CH₂O)_(m)CH₂CH₂—, or —(CH₂CH₂O)_(m)CH₂CH₂NH—;Q′ is NH, O, S, CH₂, C(O)O, C(O)NH, —NH—CH(R^(a))—C(O)—,—C(O)—CH(R^(a))—NH—, CO,

R^(a) is H or amino acid side chain; R′ and R″ are each independently H,CH3, OH, SH, NH2, NH(Alkyl) or N(diAlkyl); Z′, Z″, Z′″ and Z″″ areindependently O or S; n represent independently for each occurrence1-20; and m represent independently for each occurrence 0-50.
 3. TheiRNA agent of claim 1, wherein said iRNA agent is double stranded. 4.The iRNA agent of claim 3, wherein said monomer is at the 3′-end of oneof the strands.
 5. The iRNA agent of claim 4, wherein said monomer is atthe 3′-end of sense strand.
 6. The iRNA agent of claim 1, wherein theiRNA agent further comprises at least one monomer of formula (LI)

wherein X⁶ and Y⁶ are each independently H, a hydroxyl protecting group,a phosphate group, a phosphodiester group, an activated phosphate group,an activated phosphite group, a phosphoramidite, a solid support,—P(Z′)(Z″)O-nucleoside, —P(Z′)(Z″)O-oligonucleotide, a lipid, a PEG, asteroid, a polymer, —P(Z′)(Z″)O—R¹-Q′-R²—OP(Z′″)(Z″″)O-oligonucleotide,a nucleotide, or an oligonucleotide, —P(Z′)(Z″)-formula (I) or—P(Z′)(Z″)—; Q⁶ is absent or —(P⁶-Q⁶-R⁶)_(v)-T⁶-; P⁶ and T⁶ are eachindependently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O),CH₂, CH₂NH or CH₂O; Q⁶ is independently for each occurrence absent,substituted alkylene wherein one or more methylenes can be interruptedor terminated by one or more of O, S, S(O), SO₂, N(R^(N)), C(R′)═C(R′),C≡C or C(O); R⁶ is independently for each occurrence absent, NH, O, S,CH₂, C(O)O, C(O)NH, NHCH(R^(a))C(O), —C(O)—CH(R^(a))—NH—, CO, CH═N—O,

 or heterocyclyl; R′ and R″ are each independently H, C₁-C₆ alkyl OH,SH, N(R^(N))₂; R^(N) is independently for each occurrence methyl, ethyl,propyl, isopropyl, butyl or benzyl; R^(a) is H or amino acid side chain;Z′, Z″, Z′″ and Z″″ are each independently for each occurrence O or S; vrepresent independently for each occurrence 0-20; R^(L) is a lipophileor a cationic lipid.
 7. The RNAi agent of claim 6, wherein R^(L) is alipophile.
 8. The RNAi agent of claim 7, wherein R^(L) is cholesterol.9. The RNAi agent of claim 1, wherein R is chosen from a groupconsisting of


10. The RNAi agent of claim 1, wherein said monomer is chosen from agroup consisting of


11. The RNAi agent of claim 10, wherein R is


12. A method of modulating the expression of a target gene in a cell,comprising providing to said cell an iRNA agent of claim
 1. 13. Themethod of claim 18, wherein the target gene is selected from the groupconsisting of CD45, GFP, Factor VII, Eg5, PCSK9, TPX2, apoB, SAA, TTR,RSV, PDGF beta gene, Erb-B gene, Src gene, CRK gene, GRB2 gene, RASgene, MEKK gene, JNK gene, RAF gene, Erk1/2 gene, PCNA(p21) gene, MYBgene, JUN gene, FOS gene, BCL-2 gene, Cyclin D gene, VEGF gene, EGFRgene, Cyclin A gene, Cyclin E gene, WNT-1 gene, beta-catenin gene, c-METgene, PKC gene, NFKB gene, STAT3 gene, survivin gene, Her2/Neu gene,topoisomerase I gene, topoisomerase II alpha gene, mutations in the p73gene, mutations in the p21(WAF1/CIP1) gene, mutations in the p27(KIP1)gene, mutations in the PPM1D gene, mutations in the RAS gene, mutationsin the caveolin I gene, mutations in the MIB I gene, mutations in theMTAI gene, mutations in the M68 gene, mutations in tumor suppressorgenes, and mutations in the p53 tumor suppressor gene.
 14. Apharmaceutical composition comprising an iRNA agent of claim 1 alone orin combination with a pharmaceutically acceptable carrier or excipient.